CA2218188A1 - Solid phase sequencing of biopolymers - Google Patents

Abstract

This invention relates to methods for detecting and sequencing target nucleic acid sequences, and double-stranded nucleic acid sequences, to nucleic acid probes, to mass modified nucleic acid probes, to arrays of probes useful in these methods and to kits and systems which contain these probes. Useful methods involve hybridizing the nucleic acids or nucleic acids which represent complementary or homologous sequences of the target to an array of nucleic acid probes. These probes comprise a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. The molecular weights of the hybridized nucleic acids of the set can be determined by mass spectroscopy, and the sequence of the target determined from the molecular weights of the fragments. Nucleic acids whose sequences can be determined include DNA or RNA in biological samples such as patient biopsies and environmental samples. Probes may be fixed to a solid support such as a hybridization chip to facilitate automated molecular weight analysis and identification of the target sequence.

Description

wo 96/32504 PCT~US96/05136 SOLID PHASE SEQUENCING OF BIOPOLYMERS
~i~ht~ in the Invention This invention was made with United States Government " support under grant number DE-FG-02-93ER61609, awarded by the United S States Department of Energy, and the United States Govçrnment has certain rights in the invention.
Rack~round ofthe Invention l . Field of the Invention This invention relates to methods for ~letecting and sequenc~ng nucleic acids using sequencing by hybridization technology and molecular weight analysis. The invention also relates to probes and arrays useful in sequencing and detection and to kits and apparatus for determining sequence information.

2. Description of the Background Since the recognition of nucleic acid as the carrier of the genetic code, a great deal of interest has centered around determining the sequence of that code in the many forms which it is found. Two landmark studies made the process of nucleic acid sequencing, at least with DNA~ a common and relatively rapid procedure practiced in most laboratories. The 20 first describes a process whereby terrnin~lly labeled DNA molecules are chemically cleaved at single base repetitions (A.M. Maxam and W. Gilbert, Proc. Natl. Acad. Sci. USA 74:560-64, 1977). Each base position in the nucleic acid sequence is then determined from the molecular weights of fr~nent~ produced by partial cleavages. Individual reactions were devised 25 to cleave preferentially at guanine, at adenine, at cytosine and thymine at cytosine alone. When the products of these four reactions are resolved by molecular weight. using, for example, polyacrylamide gel electrophoresis, WO 96t32504 PCl[~/USg6/OS136 DNA sequences can be read from ~e pattern of f~gments on ~e resolved gel.
The second study describes a procedure whereby DNA is sequenced using a variation ofthe plus-minus method (F. Sanger et al., Proc.
S Natl. Acad. Sci. USA 74:5463-67, 1977). This procedure takes advantage of the chain t~l~nin~ting ability of dideoxynucleoside triphosphates (ddNTPs) and the ability of DNA polymerase to incorporate ddNTPs with nearly equal fidelity as the natural substrate of DNA polymerase, deoxynucleosides triphosphates (dNTPs). Briefly, a primer, usually an 10 oligonucleotide, and a template DNA are incubated together in the presence of a useful concentration of all four dNTPs plus a limited amount of a single ddNTP. The DNA polymerase occasionally incorporates a dideoxynucleotide which termin~tes chain extension. Because the dideoxynucleotide has no 3'-hydroxyl, the initiation point for the polymerase 15 enzyme is lost. Polymerization produces a mixture of fragments of varied sizes, all having identical 3' termini. Fractionation of the mixture by, for example, polyacrylamide gel electrophoresis, produces a pattern which indicates the presence and position of each base in the nucleic acid.
Reactions with each of the four ddNTPs allows one of ordinary skill to read 20 an entire nucleic acid sequence from a resolved gel.
Despite their advantages, these procedures are cumbersome and impractical when one wishes to obtain megabases of sequence information. Further, these procedures are, for all practical purposes, limited to sequencing DNA. Although variations have developed, it is still 25 not possible using either process to obtain sequence information directly from any other form of nucleic acid.

WO 96t32504 PC~t~JS96~(15136 A relatively new method for obtaining sequence information from a nucleic acid has recently been developed whereby the sequences of groups of c~ nti~lQus bases are determined simultaneously. In comparison to traditional techniques whereby one determines base specific inforrnation 5 of a sequence individually, this method, referred to as sequencing by hybridization (SBH), represents a many-fold amplification in speed. Due, at least in part to the increased speed, SBH presents numerous advantages including re~ ce~l expense and greater accuracy. Two general approaches of sequencing by hybridization have been suggested and their practicality 10 has been demonstrated in pilot studies. In one format, a complete set of 4"
nucleotides of length n is immobilized as an ordered array on a solid support and an unknown DNA sequence is hybridized to this array (K.R. Khrapko et al., J. DNA Sequencing and Mapping 1:375-88, 1991). The resulting hybridization pattern provides all "n-tuple" words in the sequence. This is 15 sufficient to determine short sequences except for simple tandem repeats.
In the second format, an array of immobilized samples is hybridized with one short oligonucleotide at a time (Z. Strezoska et al., Proc.
Natl. Acad. Sci. USA 88:10,089-93~ 1991). When repeated 4n times for each oligonucleotide of length n, much of the sequence of all the immobilized 20 samples would be determined. In both approaches, the intrinsic power of the method is that many sequenced regions are determined in parallel. In actual practice the array size is about 104 to 105.
Another aspect of the method is that information obtained is quite recllln(l~n~ and especially as the size of the nucleic acid probe grows.
25 Mathematical simulations have shown that the method is quite resistant to experimental errors and that far fewer than all probes are necessary to determine reliable sequence data (P.A. Pevmer et al., J. Biomol. Struc. &
Dyn. 9:399-410, 1991; W. Bains, Genomics 11:295-301,1991).
In spite of an overall optimistic outlook, there are still a number of potentially severe drawbacks to actual implementation of 5 sequencing by hybridization. First and foremost among these is that 4n rapidly becomes quite a large number if chemical synthesis of all of the oligonucleotide probes is actually contemplated. Various schemes of automating this synthesis and compressing the products into a small scale array, a sequencing chip, have been proposed.
There is also a poor level of discrimin~tion between a correctly hybridized, perfectly matched duplexes, and end mi~m~tches. In part, these drawbacks have been addressed at least to a small degree by the method of continuous stacking hybridization as reported by a Khrapko et al.
(FEBS Lett. 256:118-22, 1989). Continuous stacking hybridization is based 15 upon the observation that when a single-stranded oligonucleotide is hybridized adjacent to a double-stranded oligonucleotide, the two duplexes are mutually stabilized as if they are positioned side-to-side due to a stacking contact between them. The stability of the interaction decreases significantly as stacking is disrupted by nucleotide displacement, gap or 20 terminal mi.~m~tch Internal mi~m~tçhes are presumably ignorable because their thermodynamic stability is so much less than perfect matches.
Although promising, a related problem arises which is the inability to distinguish between weak, but correct duplex formation, and simple background such as non-specific adsorption of probes to the underlying 25 support matrix.

wo 96/32s04 PCTJU~96l05136 Detection is also monochromatic wherein separate sequential positive and negative controls must be run to discrimin~te between a correct r hybridization match, a mis-match, and background. All too often, ambiguities develop in reading sequences longer than a few hundred base pairs on account of sequence recurrences. For example, if a sequence one base shorter than the probe recurs three times in the target, the sequence position cannot be uniquely determined. The locations of these sequence ambiguities are called branch points.
Secondary structures often develop in the target nucleic acid affecting accessibility of the sequences. This could lead to blocks of sequences that are unreadable if the secondary structure is more stable than occurs on the complementary strand.
A final drawback is the possibility that certain probes will have anomalous behavior and for one reason or another, be recalcitrant to hybridization under whatever standard sets of conditions llltim~tely used.
A simple example of this is the difficulty in finding matching conditions for probes rich in G/C content. A more complex example could be sequences with a high propensity to form triple helices. The only way to rigorously explore these possibilities is to carry out extensive hybridization studies withall possible oligonucleotides of length "n" under the particular format and conditions chosen. This is clearly impractical if many sets of conditions are involved.
Among the early publication which appeared discussing sequencing by hybridization, E.M. Southern (WO 89/10977), described v 25 methods whereby unknown, or target, nucleic acids are labeled, hybridized to a set of nucleotides of chosen length on a solid support~ and the nucleotide sequence of the target ~letetTnined, at least partially, from knowledge of the sequence of the bound fragments and the pattern of hybridization observed.
Although promi~in~, as a practical matter, this method has numerous drawbacks. Probes are entirely single-stranded and binding stability is dependent upon the size of the duplex. However, every additional nucleotide of the probe necessarily increases the size of the array by four fold creating a dichotomy which severely restricts its plausible use. Further, there is an inability to deal with branch point ambiguities or secondary structure of the target, and hybridization conditions will have to be tailored or in some way accounted for each binding event. Attempts have been made to overcome or circumvent these problems.
R. Drmanac et al. (U.S. Patent No. 5,202,231) is directed to methods for sequencing by hybridization using sets of oligonucleotide probes with random or variable sequences. These probes, although useful, suffer from some of the same drawbacks as the methodology of Southern (1989), and like Southern, fail to recognize the advantages of stacking interactions.
K.R. Khrapko et al. (FEBS Lett. 256:118-22, 1989; and J.
DNA Sequencing and Mapping 1:357-88, 1991) attempt to address some of these problems using a technique referred to as continuous stacking hybridization. With continuous stacking, conceptually, the entire sequence of a target nucleic acid can be determined. Basically, the target is hybridized to an array of probes, again single-stranded, denatured from the array, and the dissociation kinetics of denaturation analyzed to determine the target sequence. Although also promising, discrimination between matches "
and mis-matches (and simple background) is low and, further, as WO 96/~S2504 PCT/US~6/05136 hybridization conditions are inconstant for each duplex, discrimin~tion becomes increasingly re~lllce-l with increasing target complexity.
Another major problem with current sequencing formats is the inability to eff1ciently detect sequence information. In conventional S procedures, individual sequences are separated by, for example, electrophoresis using capillary or slab gels. This step is slow, expensive and requires the talents of a number of highly trained individuals, and, more importantly, is prone to error. One attempt to overcome these difficulties has been to utilize the technology of mass spectrometry.
Mass spectrometry of organic molecules was made possible by the development of instruments able to volatize large varieties of organic compounds and by the discovery that the molecular ion forrned by volatization breaks down into charged fragments whose structures can be related to the intact molecule. Although the process itself is relatively straight forward, actual implementation is quite complex. Briefly, the sample molecule or analyte is volatized and the resulting vapor passed into an ion chamber where it is bombarded with electrons accelerated to a compatible energy level. Electron bombardment ionizes the molecules of the sample analyte and then directs the ions formed to a mass analyzer. The mass analyzer, with its combination of electrical and magnetic fields, separates impacting ions according to their mass/charge (m/e) ratios. From these ratios, the molecular weights of the impacting ions can be determined and the structure and molecular weight of the analyte deterrnined. The entire process requires less than about 20 microseconds.
Attempts to apply mass spectrometry to the analysis of biomolecules such as proteins and nucleic acids have been disappointing.

Mass spectrometric analysis has traditionally been limit~d to molecules with molecular weights of a few ~ousand ~ ton~. At higher molecular weights, samples become increasingly difficult to volatize and large polar molecules generally cannot be vaporized without catastrophic consequences. The 5 energy requirement is so significant that the molecule is destroyed or, even worse, fragmented. Mass spectra of fragmented molecules are often difficult or impossible to read. Fragment linking order, particularly useful for reconstructing a molecular structure, has been lost in the fragmentation process. Both signal to noise ratio and resolution are significantly 10 negatively affected. In addition, and specifically with regard to biomolecular sequencing, extreme sensitivity is necessary to detect the single base differences between biomolecular polymers to determine sequence identity.
A number of new methods have been developed based on the 15 idea that heat, if applied with sufficient rapidity, will vaporize the samplebiomolecule before decomposition has an opportunity to take place. This rapid heating technique is referred to as plasma desorption and there are many variations. For example, one method of plasma desorption involves placing a radioactive isotope such as Californium-252 on the surface of a 20 sample analyte which forms a blob of plasma. From this plasma, a few ions of the sample molecule will emerge intact. Field desorption ionization, another form of desorption, utilizes strong electrostatic fields to literally extract ions from a substrate. In secondary ionization mass spectrometry or fast ion bombardment, an analyte surface is bombarded with electrons which 25 encourage the release of intact ions. Fast atom bombardment involves bombarding a surface with accelerated ions which are neutralized by a charge exchange before they hit the surface. Presumably, neutralization of the charge lessens the probability of molecular destruction, but not the creation of ionic forms of the sample. In laser desorption, photons comprise the vehicle for depositing energy on the surface to volatize and ionize molecules of the sample. Each of these techniques has had some measure of success with different types of sample molecules. Recently, there have also been a variety of techniques and combinations of techniques specifically directed to the analysis of nucleic acids.
Brennan et al. used nuclide markers to identify terminal nucleotides in a DNA sequence by mass speckometry (U.S. Patent No.
5,003,059). Stable nuclides, ~ietect~ble by mass spectrometry, were placed in each ofthe four dideoxynucleotides used as reagents to polymerize cDNA
copies of the target DNA sequence. Polymerized copies were separated electrophoretically by size and the terrninal nucleotide identified by the presence of the unique label.
Fenn et al. describes a process for the production of a mass spectrum cont~ining a multiplicity of peaks (U.S. Patent No. 5,130,538).
Peak components comprised multiply charged ions formed by dispersing a solution containing an analyte into a bath gas of highly charged droplets.
An electrostatic field charged the surface of the solution and dispersed the liquid into a spray referred to as an electrospray (ES) of charged droplets.
This nebulization provided a high charge/mass ratio for the droplets increasing the upper limit of volatization. Detection was still limited to less than about 100,000 daltons.
Jacobson et al. utilizes mass spectrometry to analyze a DNA
sequence by incorporating stable isotopes into the sequence (U.S. Patent No.

5,002,868). Incorporation required the steps of enzymatically introducing the isotope into a strand of DNA at a terminus, electrophoretically s~a,dling the strands to determine fragment size and analyzing the separated strand by mass spectrometry. Although accuracy was stated to 5 have been increased, electrophoresis was necessary to isolate the labeled strand.
Brennan also utilized stable markers to label the terminal nucleotides in a nucleic acid sequence, but added the step of completely degrading the components of the sample prior to analysis (U.S . Patent Nos.
10 5,003,059 and 5,174,962). Nuclide markers, enzymatically incorporated into either dideoxynucleotides or nucleic acid primers, were eleckophoretically separated. Bands were collected and subjected to combustion and passed through a mass spectrometer. Combustion converts the DNA into oxides of carbon, hydrogen, nitrogen and phosphorous, and 15 the label into sulfur dioxide. Labeled combustion products were identified and the mass of the initial molecule reconstructed. Although fairly accurate, the process does not lend itself to large scale sequencing of biopolymers.
A recent advancement in the mass spectrometric analysis of high molecular weight molecules in biology has been the development of 20 time of flight mass spectrometry (TOF-MS) with matrix-assisted laser desorption ionization (MALDI). This process involves placing the sample into a matrix which contains molecules which assist in the desorption process by absorbing energy at the frequency used to desorp the sample.
The theory is that volatization of the matrix molecules encourages 25 volatization of the sample without significant destruction. Time of flight analysis utilizes the travel time or flight time of the various ionic species as WQ 96132504 PCTlUSg6~()S136 an accurate indicator of molecular mass. There have been some notable successes with these techniques.
Beavis et al. proposed to measure the molecular weights of DNA fr~rnent~ in mixtures prepared by either Maxam-Gilbert or Sanger 5 sequencing techniques (U.S. Patent No. 5,288,644). Each of the different DNA fr~ t~ to be generated would have a common origin and tçrrnin~te at a particular base along an unknown sequence. The separate mixtures would be analyzed by laser desorption time of flight mass spectroscopy to deterrnine fr~nent molecular weights. Spectra obtained from each reaction 10 would be compared using computer algc~liLhllls to determine the location of each of the four bases and ultimately, the sequence of the fragment.
Williams et al. utilized a combination of pulsed laser ablation7 multiphoton ionization and time of flight mass spectrometry. Effective laser desorption was accomplished by ablating a frozen film of a solution 15 containing sample molecules. When ablated, the film produces an expanding vapor plume which entrains the intact molecules for analysis by mass spectrometry.
Even more recent developments in mass spectrometry have further increased the upper limits of molecular weight detection and 20 determination. Mass spectrograph systems with reflectors in the flight tube have effectively doubled resolution. Reflectors also compensate for errors in mass caused by the fact that the ionized/accelerated region of the instrument is not a point source, but an area of finite size wherein ions can accelerate at any point. Spatial differences between particle the origination 25 points of the particles, problematic in conventional instruments because arrival times at the detector will vary, are overcome. Particles that spend more time in the accelerating field will also spend more time in the Lcla.ding field. Therefore, particles emerging from the reflector are mostly synchronous, vastly improving resolution.
Despite these advances, it is still not possible to generate 5 coordinated spectra representing a continuous sequence. Furthermore, throughput is sufflciently slow so as to make these methods impractical for large scale analysis of sequence information.

Sllmm~ of tlle Jnvention The present invention overcomes the problems and disadvantages associated with current strategies and designs and provides methods, kits and apparatus for determining the sequence of target nucleic aclds.
One embodiment of the invention is directed to methods for 15 sequencing a target nucleic acid. A set of nucleic acid fragments containing a sequence which is complementary or homologous to a sequence of the target is hybridized to an array of nucleic acid probes wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, forming a target array 20 of nucleic acids. Molecular weights for a plurality of nucleic acids of the target array are determined and the sequence of the target constructed.
Nucleic acids of the target, the target sequence, the set and the probes may be DNA, RNA or PNA comprising purine, pyrimidine or modified bases.
The probes may be fixed to a solid support such as a hybridization chip to 25 facilitate automated determination of molecular weights and identification of the target sequence.

WO 96/32504 PCTIUS96~05136 Another embodiment of the invention is directed to methods for sequencing a target nucleic acid. A set of nucleic acid fr~rnent.c cont~inin~ a sequence which is complem~nt~ry or homologous to a sequence of the target is hybridized to an array of nucleic acid probes S forming a target array cont~inin ~ a plurality of nucleic acid complexes. One strand of those probes hybridized by a fragment is extended using the fr~grnent as a template. Molecular weights for a plurality of nucleic acids ofthe target array are ~let~rmined and the sequence ofthe target constructed.
Strands can be enzymatically extended using chain termin~ting and chain elon~ting nucleotides. The resulting nested set of nucleic acids represents the sequence of the target.
Another embodiment of the invention is directed to methods for detecting a target nucleic acid. A set of nucleic acids complementary to a sequence of the target, is hybridized to a fixed array of nucleic acid probes.The molecular weights of the hybridized nucleic acids are determined by mass spectrometry and a sequence of the target can be identified. Target nucleic acids may be obtained from biological samples such as patient samples wherein detection of the target is indicative of a disorder in the patient, such as a genetic defect, a neoplasm or an infection.
Another embodiment of the invention is directed to methods for sequencing a target nucleic acid. A sequence of the target is cleaved into nucleic acid fragrnents and the fragments hybridized to an array of nucleic acid probes. Fr~gment~ are created by enzymatically or physically cleaving the target and the sequence of the fragments is homologous with or v 25 complementary to at least a portion of the target sequence. The array is attached to a solid support and the molecular weights of the hybridized fr~nentr~ det~rmined by mass spectrometry. From the molecular weights d~t~rmined, nucleotide sequences of the hybridized fragments are ~letçrmined and a nucleotide sequence of the target can be identified.
Another embodiment of the invention is directed to methods S for sequencing a target nucleic acid. A set of nucleic acids complementary to a sequence of the target is hybridized to an array of single-stranded nucleic acid probes wherein each probe comprises a constant sequence and a variable sequence and said variable sequence is determinable. The molecular weights of the hybridized nucleic acids are determined and the 10 sequence of said target identified. The array comprises less than or equal toabout 4R different probes and R is the length in nucleotides of the variable sequence and may be attached to a solid support.
Another embodiment of the invention is directed to methods for sequencing a target nucleic acid by strand-displacement, double-stranded lS sequencing. A set of partially single-stranded and partially double-stranded nucleic acid fragments are provided wherein each fragment contains a sequence that corresponds to a sequence of the target. These nucleic acid fragments are hybridized to a set of partially single-stranded and partially double-stranded nucleic acid probes, via the single-stranded regions of each, 20 to form a set of fragment/probe complexes. Prior to hybridization, either the fragments or the probes may be treated with a phosphorylase to remove phosphate groups from the 5'-termini of the nucleic acids. 5'-termini are ligated with adjacent 3'-termini of the complex forming a common single strand. The complementary unligated strand contains a nick which is 25 recognized by a nucleic acid polymerase that initiates strand-displacement polymerization~ extending the unligated strand. Polymerization proceeds, WO 96/32504 PCI'IUS961(~5136 using the ligated strand as a template, in the presence of labeled nucleotides such as mass modified nucleotides. The sequence of the target can be ~leterrnined by mass spectrometry from the molecular weights of the ex~n~ecl strands. This process can be used to sequence target nucleic acids 5 and also to identify a single sequence in a mixed background. Selection of the species of nucleic acid to be sequenced occurs upon hybridization to the probe. As only fragments complementary to the single-stranded region of the probe will form complexes, only those fragments complexes are sequenced.
Another embodiment of the invention is directed to arrays of nucleic acid probes. In these arrays, each probe comprises a first strand and a second strand wherein the first strand is hybridized to the second strand forrning a double-stranded portion, a single-stranded portion and a variable sequence within the single-stranded portion. The array may be attached to 15 a solid support such as a material that facilitates volatization of nucleic acids for mass spectrometry. Arrays can be fixed to hybridization chips cont~ining less than or equal to about 4R different probes wherein R is the leng~th in nucleotides of the variable sequence. Arrays can be used in detection methods and in kits to detect nucleic acid sequences which may 20 be indicative of a disorder and in sequencing systems such as sequencing by mass spectrometry.
Another embodiment of the invention is directed to arrays of single-stranded nucleic acid probes wherein each probe of the arra~
comprises a constant sequence and a variable sequence which is 25 determinable. Arrays may be attached to solid supports which comprise matrices that facilitate volatization of nucleic acids for mass spectrometry.

Arrays, generated by conventional processes, may be characterized using the above methods and replicated in mass for use in nucleic acid detection and sequencing systems.
Another embodiment of the invention is directed to kits for S ~letecting a sequence of a target nucleic acid. Kits contain arrays of nucleic acid probes fixed to a solid support wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion. The solid support may be, for example, coated with a matrix that facilitates volatization of nucleic acids for mass 10 spectrometry such as an aqueous composition.
Another embodiment of the invention is directed to mass spectrometry systems for the rapid sequencing of nucleic acids. Systems comprise a mass spectrometer, a computer with ay~lopliate software and probe arrays which can be used to capture and sort nucleic acid sequences 15 for subsequent analysis by mass spectrometry.
Other embodiments and advantages of the invention are set forth, in part, in the description which follows and, in part, will be obvious from this description and may be learned from the practice of the invention.

20 De~cription of the Drawin~.c Figure 1 (A) Schematic of a mass modified nucleic acid primer; and (B) primer mass modification moieties.
Figure 2 (A) Schematic of mass modified nucleoside triphosphate elongators and terminators; and (B) nucleoside triphosphate mass modification moieties.
Figure 3 List of mass modification moieties.

WO 96132504 PCTlUS96rO5136 Figure 4 List of mass modification moieties.
Figure 5 Cleavage site of Mwo 1 indicating bidirectional sequencing.
Figure 6 Sehem~tic of seql7encin~ strategy after target DNA digestion by ~sp Rl.
Figure 7 Calc~ te~l Tm of matched and mi~m~tçhed complement~ry DNA.
Figure 8 Replication of a master array.
Figure 9 Reaction scheme for the covalent attachment of DNA to a surface.
Figure 10 Target nucleic acid capture and ligation.
Figure 11 Ligation efficiency of matches as compared to mi~m~tches.
Figure 12 (A) Ligation of target DNA with probe attached at 5'-terrninlls; and (B) ligation of target DNA with probe attached at the 3'-terminus.
Figure 13 Gel reader sequencing results from primer hybridization analysis.
Figure 14 Mass spectrometry of oligonucleotide ladder.
Figure 15 Schematic of mass modification by alkylation.
Figure 16 Mass spectrum of 1 7-mer target with 0, 1 or 2 mass modified moieties.
- Figure 17 Schematic of nicked strand displacement sequencing with immobilized template.
Figure 18 Analysis of sequencing reaction in the presence and absence of single-stranded DNA binding protein.
Figure 19 Schematic of nicked strand displacement sequencing with immobilized probe.

WO 96/32504 PCTrUS96/05136 Figure 20 Results of sequencing performed using DF27- 1 as a probe.
Figure 21 Results of sequencing performed using DF27-2 as a probe.
Figure 22 Results of sequencing performed using DF27-4 as a probe.
Figure 23 Results of sequencing ~ rolmed using DF27-5-CY5 as a probe.
Figure 24 Results of sequencing performed using DF27-6-CY5 as a probe.

nescription of the Invention As embodied and broadly described herein, the present invention is directed to methods for sequencing a nucleic acid, probe arrays useful for sequencing by mass spectrometry and kits and systems which comprise these arrays.
Nucleic acid sequencing, on both a large and small scale, is 15 critical to many aspects of medicine and biology such as, for example, in theidentification~ analysis or diagnosis of diseases and disorders, and in determining relationships between living org~ni.sms. Conventional sequencing techniques rely on a base-by-base identification of the sequence using electrophoresis in a semi-solid such as an agarose or polyacrylamide 20 gel to determine sequence identity. Although attempts have been made to apply mass spectrometric analysis to these methods, the two processes are not well suited because, at least in part, information is still be gathered in asingle base format. Sequencing-by-hybridization methodology has enhanced the sequencing process and provided a more optimistic outlook for 25 more rapid sequencing techniques, however, this methodology is no more applicable to mass spectrometry than traditional sequencing techniques.

WO 96t32504 PCT~US9610SI36 In contrast, positional sequencing by hybridization (PSBH) with its ability to stably bind and discrimin~te different sequences with large O or small arrays of probes is well suited to mass spectrometric analysis.
Sequence information is rapidly ~letermined in batches and with a minimllm 5 of effort. Such processes can be used for both sequencing unknown nucleic acids and for detecting known sequences whose presence may be an indicators of a disease or con~ tion. Additionally, these processes can be lltili7e~1 to create coortlin~te~l patterns of probe arrays with known sequences. Determination of the sequence of fragments hybridized to the 10 probes also reveals the sequence ofthe probe. These processes are currently not possible with conventional techniques and, further, a coor lin~tecl batch-type analysis provides a significant increase in sequencing speed and accuracy which is expected to be required for effective large scale sequenclng operatlons.
PSBH is also well suited to nucleic acid analysis wherein sequence information is not obtained directly from hybridization. Sequence information can be learned by coupling PSBH with techniques such as mass spectrometry. Target nucleic acid sequences can be hybridized to probes or array of probes as a method of sorting nucleic acids having distinct 20 sequences without having a priori knowledge of the sequences of the various hybridization events. As each probe will be represented as multiple copies, it is only necessary that hybridization has occurred to isolate distinctsequence packages. In addition, as distinct packages of sequences, they can be amplified, modified or otherwise controlled for subsequent analysis.
25 Amplification increases the number of specific sequences which assists in any analysis requiring increased quantities of nucleic acid while retainin sequence specificity. Modif1cation may involve chemically altering the nucleic acid molecule to assist with later or downstream analysis.
Consequently, another important feature ofthe invention is the ability to simply and rapidly m~s modify the sequences of interest. A mass 5 modification is an alteration in the mass, typically measured in terms of molecular weight as daltons, of a molecule. Mass modification which increase the discrimin~ion between at least two nucleic acids with single base differences in size or sequence can be used to facilitate sequencing using, for example, molecular weight determinations.
One embodiment of the invention is directed to a method for sequencing a target nucleic acid using mass modified nucleic acids and mass spectrometry technology. Target nucleic acids which can be sequenced include sequences of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Such sequences may be obtained from biological, recombinant or 15 other man-made sources, or purified from a natural source such as a patient'stissue or obtained from environmental sources. Alternate types of molecules which can be sequenced includes polyamide nucleic acid (PNA) (P.E.
Nielsen et al., Sci. 254:1497-1500, 1991) or any sequence of bases joined by a chemical backbone that have the ability to base pair or hybridize with 20 a complementary chemical structure.
The bases of DNA, RNA and PNA include purines, pyrimidines and purine and pyrimidine derivatives and modifications, which are linearly linked to a chemical backbone. Common chemical backbone structures are deoxyribose phosphate, ribose phosphate, and polyamide. The 25 purines of both DNA and RNA are adenine (A) and guanine (G). Others that are known to exist include xanthine, hypoxanthine. 2- and l-WO ~6132504 PCTIUS~?6~(~5136 ~ minopurine, and other more modified bases. The pyrimidines are cytosine (C), which is common to both DNA and RNA, uracil (U) found - pre~lomin~ntly in RNA, and thymidine (T) which occurs almost exclusively in DNA. Some of the more atypical pyrimidines include methylcytosine, hydroxymethyl-cytosine, methyluracil, hydroxymethyluracil, dihydroxypentyluracil, and other base modifications. These bases interact in a complementary fashion to form base-pairs, such as, for example, guanine with cytosine and adenine with thymidine. This invention a~so encompasses situations in which there is non-traditional base pairing such as Hoogsteen base pairing which has been identified in certain tRNA
molecules and postulated to exist in a kiple helix.
Sequencing involves providing a nucleic acid sequence which is homologous or complementary to a sequence of the target. Sequences may be chemically synthesized using, for example, phosphoramidite chemistry or created enzymatically by incubating the target in an a~pr~liate buffer with chain elongating nucleotides and a nucleic acid polymerase.
Initiation and termination sites can be controlled with dideoxynucleotides or oligonucleotide primers, or by placing coded signals directly into the nucleic acids. The sequence created may comprise any portion of the target sequence or the entire sequence. Alternatively, sequencing may involve elongating DNA in the presence of boron derivatives of nucleotide kiphosphates. Resulting double-stranded samples are treated with a 3' exonuclease such as exonuclease III. This exonuclease stops when it encounters a boronated residue thereby creating a sequencing ladder.
Nucleic acids can also be purified, if necessary to remove substances which could be harmful (e.g. toxins), dangerous (e.g. infectious) CA 022l8l88 l997- lO- l4 WO g6/32504 PCTIUS96/OS136 or might interfere with the hybridization reaction or the sensitivity of that reaction (e.g metals, salts, protein, lipids). Purification may involve techniques such as chemical extraction with salts, chloroform or phenol, se-liment~tion centrifugation, chromatography or other techniques known 5 to those of ordinary skill in the art.
If sufficient quantities of target nucleic acid are available and the nucleic acids are sufficiently pure or can be purified so that any substances which would interfere with hybridization are removed, a plurality of target nucleic acids may be directly hybridized to the array. Sequence 10 information can be obtained without creating complementary or homologous copies of a target sequence.
Sequences may also be amplified, if necessary or desired, to increase the number of copies of the target sequence using, for example, polymerase chain reactions (PCR) technology or any of the amplification 15 procedures. Amplification involves denaturation of template DNA by heating in the presence of a large molar excess of each of two or more oligonucleotide primers and four dNTPs (dGTP, dCTP, dATP, dTTP). The reaction mixture is cooled to a temperature that allows the oligonucleotide primer to anneal to target sequences, after which the annealed primers are 20 ext~nded with DNA polymerase. The cycle of denaturation, annealing, and DNA synthesis, the principal of PCR amplification, is repeated many times to generate large quantities of product which can be easily identified.
The major product of this exponential reaction is a segment of double stranded DNA whose termini are defined by the 5' termini of the 25 oligonucleotide primers and whose length is defined by the distance between the primers. Under normal reaction conditions, the amount of polymerase WO 96132504 PCI'IUS9610S136 becomes limiting after 25 to 30 cycles or about one million fold amplification. Further, amplification is achieved by diluting the sample 1000 fold and using it as the template for further rounds of amplification in another PCR. By this method, amplification levels of 109 to 10'~ can be S achieved during the course of 60 sequential cycles. This allows for the detection of a single copy of the target sequence in the presence of Co~ ";"~tin~ DNA, for example, by hybridization with a radioactive probe.
With the use of sequential PCR, the practical detection limit of PCR can be as low as 10 copies of DNA per sample.
Although PCR is a reliable method for amplification of target sequences, a number of other techniques can be used such as ligase chain reaction, self sustained sequence replication, Q,B replicase amplification, polymerase chain reaction linked ligase chain reaction, gapped ligase chain reaction, ligase chain detection and strand displacement amplification. The 1~ principle of ligase chain reaction is based in part on the ligation of two adjacent synthetic oligonucleotide primers which uniquely hybridize to one strand of the target DNA or RNA. If the target is present, the two oligonucleotides can be covalently linked by ligase. A second pair of primers, almost entirely complementary to the first pair of primers is also 20 provided. The template and the four primers are placed into a thermocycler with a thermostable ligase. As the temperature is raised and lowered, oligonucleotides are renatured immediately adjacent to each other on the template and ligated. The ligated product of one reaction serves as the template for a subsequent round of ligation. The presence of target is 2~ manifested as a DNA fragment with a length equal to the sum of the two adjacent oligonucleotides.

Target sequences are fr~rnent~.l, if necç~s~ry, into a plurality of fragments using physical, chemical or enzymatic means to create a set of fragments of uniform or relatively uniform length. Preferably, the sequences are enzymatically cleaved using nucleases such as DNases or S RNases (mung bean nuclease, micrococcal nuclease, DNase I, RNase A, RNase Tl), type I or II restriction endonucleases, or other site-specific or non-specific endonucleases. Sizes of nucleic acid fragments are between about 5 to about 1,000 nucleotides in length, preferably between about 10 to about 200 nucleotides in length, and more preferably between about 12 1 0 to about l OO nucleotides in length. Sizes in the range of about 5, 1 0,12,1 5, 18, 20, 24, 26, 30 and 35 are useful to perform small scale analysis of short regions of a nucleic acid target. Fragment sizes in the range of 25, 50, 75, 125, 150, 175, 200 and 250 nucleotides and larger are useful for rapidly analyzing larger target sequences.
Target sequences may also be enzymatically synthesized using, for example, a nucleic acid polymerase and a collection of chain elongating nucleotides (NTPs, dNTPs) and limiting amounts of chain terrnin~ting (ddNTPs) nucleotides. This type of polymerization reaction can be controlled by varying the concentration of chain termin~ting nucleotides 20 to create sets, for example nested sets, which span various size ranges. In a nested set, fragments will have common one terminus and one terminus which will be different between the members of the set such that the larger fragments will contain the sequences of the smaller fragments.
The set of fragments created, which may be either homologous 25 or complementary to the target sequence, is hybridized to an array of nucleicacid probes forming a target array of nucleic acid probe/fragrnent WO 96/32504 PCTJUS96~0S136 complexes. An array con~titllte~ an ordered or structuredplurality of nucleic acids which may be fixed to a solid support or in liquid suspension.
. Hybridization of the fr~nen~ to the array allows for sorting of very large eolleetions of nueleie aeid fr~ment~ into i~l~ntifi~ble groups. Sorting does S not require a priori knowledge of the sequences of the probes, and can greatly facilitate analysis by, for example, mass spectrophotometric techniques.
Hybridization between complementary bases of DNA, RNA, PNA, or combinations of DNA, RNA and PNA, occurs under a wide variety 10 of conditions such as variations in tempc-~lule, salt concentration, electrostatic strength, and buffer composition. Exarnples ofthese conditions and methods for applying them are described in Nucleic Acid Hybridizafion:
A Practical Approach (B.D. Hames and S.J. Higgins, editors, IRL Press, 1985). It is preferred that hybridization takes place between about 0~C and 15 about 70~C, for periods of from about one minute to about one hour, depending on the nature of the sequence to be hybridized and its length.
However, it is recognized that hybridizations can occur in seconds or hours, depending on the conditions of the reaction. For example, typical hybridization conditions for a mixture of two 20-mers is to bring the mixture 20 to 68~C and let cool to room temperature (22~C) for five minutes or at very low temperatures such as 2~C in 2 microliters. Hybridization between nucleic acids may be facilitated using buffers such as Tris-EDTA (TE), Tris-HCI and HEPES, salt solutions (e.g. NaCI, KCI, CaC12), other aqueous solutions, reagents and chemicals. Examples of these reagents include 25 single-stranded binding proteins such as Rec A protein, T4 gene 32 protein, E. coli single-stranded binding protein and major or minor nucleic acid WO 96/3250~ PCT/US9~i/05136 groove binding proteins. Examples of other reagents and chemicals include divalent ions, polyvalent ions and interc~l~tin~ substances such as ethidium bromide, actinomycin D, psoralen and angelicin.
Optionally, hybridized target sequences may be ligated to a 5 single-strand of the probes thereby creating ligated target-probe complexes or ligated target arrays. Ligation of target nucleic acid to probe increases fidelity of hybridization and allows for incorrectly hybridized target to be easily washed from correctly hybridized target. More importantly, the addition of a ligation step allows for hybridizations to be performed under 10 a single set of hybridization conditions. Variation of hybridization conditions due to base composition are no longer relevant as nucleic acids with high A/T or G/C content ligate with equal efficiency. Consequently, discrimination is very high between matches and mis-matches, much higher than has been achieved using other methodologies wherein the effects of 15 G/C content were only somewhat neutralized in high concentrations of quaternary or tertiary amines such as, for example, 3M tetramethyl ammonium chloride. Further, hybridization conditions such as temperatures of between about 22~C to about 37~C, salt concentrations of between about 0.05 M to about 0.5 M, and hybridization times of between about less than 20 one hour to about 14 hours (overnight), are also suitable for ligation.
Ligation reactions can be accomplished using a eukaryotic derived or a prokaryotic derived ligase such as T4 DNA or RNA ligase. Methods for use of these and other nucleic acid modif~ing enzymes are described in Current Protocols in Molecular Biology (F.M. Ausubel et al., editors, John Wiley &
25 Sons, 1989).

Each probe of the probe array comprises a single-stranded portion, an optional double-stranded portion and a variable sequence within the single-stranded portion. These probes may be DNA, RNA, PNA, or any combination thereof, and may be derived from natural sources or 5 recombinant sources, or be organically syrltl-esi7e~1 Preferably, each probe has one or more double stranded portions which are about 4 to about 30 nucleotides in length, preferably about 5 to about 15 nucleotides and more preferably about 7 to about 12 nucleotides, and may also be identical within the various probes of the array, one or more single stranded portions which 10 are about 4 to 20 nucleotides in length, preferably between about 5 to about 12 nucleotides and more preferably between about 6 to about 10 nucleotides, and a variable sequence within the single stranded portion which is about 4 to 20 nucleotides in length and preferably about 4, 5, 6, 7 or 8 nucleotides in length. Overall probe sizes may range from as small as 8 nucleotides in 15 lengths to 100 nucleotides and above. Preferably, sizes are from about 12 to about 35 nucleotides, and more preferably, from about 12 to about 25 nucleotides in length.
Probe sequences may be partly or entirely known, determinable or completely unknown. Known sequences can be created, for 20 example, by chemically synthesizing individual probes with a specified sequence at each region. Probes with determinable variable regions may be chemically synthesized with random sequences and the sequence information determined separately. Either or both the single-stranded and the double-stranded regions may comprise constant sequences such as, for 25 example, when an area of the probe or hybridized nucleic acid would benefit from having a constant sequence as a point of rc;fe~ ce in subsequent analyses.
An advantage of this type of probe is in its structure.
Hybridization of the target nucleic acid is encouraged due to the favorable 5 thermodynamic conditions, including base-stacking interactions, established by the presence of the adjacent double strandedness of the probe. Probes may be structured with t~rmin~l single-stranded regions which consist entirely or partly of variable sequences, internal single-skanded regions which contain both constant and variable regions, or combinations of these 10 structures. Preferably, the probe has a single-stranded region at one terminus and a double-stranded region at the opposite terminus.
Fragmented target sequences, preferably, will have a distribution of terminal sequences sufficiently broad so that the nucleotide sequence ofthe hybridized frS~grnçntc will include the entire sequence ofthe 15 target nucleic acid. Consequently, the typical probe array will comprise a collection of probes with sufficient sequence diversity in the variable regions to hybridize, with complete or nearly complete discrimination, all of the target sequence or the target-derived sequences. The resulting target array will comprise the entire target sequence on strands of hybridized 20 probes. By way of example only, if the variable portion consisted of a four nucleotide sequence (R=4) of adenine, guanine, thymine, and cytosine, the total number of possible combinations (4R) would be 44 or 256 different nucleic acid probes. If the number of nucleotides in the variable sequence was five, the number of different probes within the set would be 45 or 1,024.
25 In addition, it is also possible to utilize probes wherein the variable nucleotide sequence contains gapped segrnents, or positions along the W~ 96/32504 PCT(U~i96105136 variable sequence which will base pair with any nucleotide or at least not interfere with adjacent base pairing.
A nucleic acid strand of the target array may be extt?ncle~l or elongated enzymatically. Either the hybridized fr~grnent or one or the other 5 ofthe probe strands can be e~tPn-1e~1 Extension reactions can utilize various regions of the target array as a template. For example, when fr~nent sequences are longer than the hybridizable portion of a probe having a 3' single-stranded terminus, the probe will have a 3' overhang and a 5' overhang after hybridization of the fragment. The now internal 3' terminus 10 of the one strand of the probe can be used as a primer to prime an extension reaction using, for example, an a~lo~liate nucleic acid polymerase and chain elon~ting nucleotides. The extended strand of the probe will contain sequence information ofthe entire hybridized fr~nent Reaction mixtures cont~ining dideoxynucleotides will create a set of extended strands of 15 varying lengths and, preferably, a nested set of strands. As the fragments have been initially sorted by hybridization to the array, each probe of the array will contain sets of nucleic acids that represent each segment of the target sequence. Base sequence information can be determined from each extended probe. Compilation of the sequence information from the array, 20 which may require computer assistance with very large arrays, will allow one to ~et~rrnine the sequence of the target. Depending on the structure of the probe (e.g 5' overhang, 3' overhang, internal single-stranded region), strands of the probe or strands of hybridized nucleic acid containing target sequence can also be enzymatically amplified by, for example, single primer 25 PCR reactions. Variations of this process may involve aspects of strand displacement amplification, Q~ replicase amplification, self-sustained sequence replication amplification and any of the various polymerase chain reaction amplification technologies.
Fxt~n~led nucleic acid strands of the probe can be mass modified using a variety of techniques and methodologies. The most S straight forward may be to erLzymatically synthesize the extension lltili7ing a polymerase and nucleotide reagents, such as mass modified chain elongating and chain termin~ing nucleotides. Mass modified nucleotides incorporate into the growing nucleic acid chain. Mass modifications may be introduced in most sites of the macromolecule which do not interfere 10 with the hydrogen bonds required for base pair formation during nucleic acid hybridization. Typical modifications include modification of the heterocyclic bases, modifications of the sugar moiety (ribose or deoxyribose), and modifications of the phosphate group. Specifically, a modifying functionality, which may be a chemical moiety, is placed at or 15 covalently coupled to the C2, N3, N7 or N8 positions of purines, or the N7 or N9 positions of deazapurines. Modifications may also be placed at the C5 or C6 positions of pyrimidines (e.g Figures lA, lB, 2A and 2B).
Examples of useful modifying groups include deuterium, F, Cl, Br, I, biotin, fluorescein, iododicarbocyanine dye, SiR, Si(CH3)3, Si(CH3)2(C2Hs), 20 Si(CH3)2(C2Hs)2, Si(CH )~C H ~ ,5 2Si(C H ) ~ (Ç~ ) CH, 2 ~CH )3NR, 2 n CH2CONR, (CH2)nOH, CH2F, CHF2 and CF3; wherein n is an integer and R
is selected from the group consisting of-H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated 25 silyl, hetero-oligo/polyaminoacid and polyethylene glycol (Figures 3 and 4).

Mass modifying functionalities may also be generated from a precursor functionality such as -N3 or -XR, wherein X is: -OH, -NH2, -., NHR,-SH,-NCS,-OCO(CH2)nCOOH,-NHCO(CH2)nCOOH,-OSO20H, -OCO(CH2)nI or -OP(O-alkyl)-N-(alkyl)2, and n is an integer from 1 to 20;
5 and R is: -H, deuterium and alkyls, alkoxys or aryls of 1-6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, t-butyl, hexyl, benzyl, benzhydral, trityl, substituted trityl, aryl, substituted aryl, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid or polyethylene glycol.
10 These and other mass modifying functionalities which do not interfere with hybridization can be attached to a nucleic acids either alone or in combination. Preferably, combinations of different mass modifications are utilized to maximize distinctions between nucleic acids having different sequences.
1~ Mass modifications may be major changes of molecular weight, such as occurs with coupling between a nucleic acid and a heterooligo/polyaminoacid, or more minor such as occurs by substituting chemical moieties into the nucleic acid having molecular masses smaller than the natural moiety. Non-essential chemical groups may be elimin~ted 20 or modified using, for example, an alkylating agent such as iodoacetamide.
Alkylation of nucleic acids with iodo~cet~mide has an additional advantage that a reactive oxygen of the 3'-position of the sugar is elimin~ted. This provides one less site per base for alkali cations, such as sodium, to interact.Sodium, present in nearly all nucleic acids, increases the likelihood of 25 forming satellite adduct peaks upon ionization. Adduct peaks appear at a slightly greater mass than the true molecule which would greatly reduce the accuracy of molecular weight determinations. These problems can be addressed, in part, with matrix selection in mass spectrometric analysis, but this only helps with nucleic acids of less than 20 nucleotides. Ammonium (+NH3), which can substitute for the sodium cation (+Na) during ion 5 exchange, does not increase adduct forrnation. Consequently, another useful mass modification is to remove alkali cations from the entire nucleic acid.
This can be accomplished by ion exchange with aqueous solutions of arnmonium such as ammonium ~cet~te, ammonium carbonate, diammonium hydrogen citrate, ammonium tartrate and combinations of these solutions.
10 DNA dissolved in 3 M aqueous ammonium hydroxide neutralizes all the acidic functions of the molecule. As there are no protons, there is a significant reduction in fragmentation during procedures such as mass spectrometry.
Another mass modification is to utilize nucleic acids with non-15 ionic polar phosphate backbones (e.g. PNA). Such nucleotides can begenerated by oligonucleoside phosphomonothioate diesters or by enzymatic synthesis using nucleic acid polymerases and alpha- (~-) thio nucleoside triphosphate and subsequent alkylation with iodo~cet~mide. Synthesis of such compounds is straight forward and can be performed and the products 20 separated and isolated by, for example, analytical HPLC.
Mass modification of arrays can be performed before or after target hybridization as the modification do not interfere with hybridization of or hybridized nucleic. This conditioning of the array is simply to perform and easily adaptable in bulk. Probe arrays can therefore be synthesized with 2~ no special manipulations. Only after the arrays are fixed to solid supports, -WO 96/32504 PCItUS96105136 just in fact when it would be most convenient to perform mass modification, would probes be conditioned.
Probe strands may also be mass modified subsequent to synthesis by, for example, contacting by treating the extended strands with S an alkylating agent, a thiolating agent or subjecting the nucleic acid to cation exchange. Nucleic acid which can be modified include target sequences, probe sequences and strands, extended strands of the probe and other available fragment~. Probes can be mass modified on either strand prior to hybridization. Such arrays of mass modified or conditioned nucleic acids 10 can be bound to fr~rnent~ cont~inin~ the target sequence with no il~L~lr~lc,lce to the fidelity of hybridization. Subsequent extension of either strand of the probe, for example using Sanger sequencing techniques, and using the target sequences as templates will create mass modified extended strands. ~he molecular weights of these strands can be determined with 15 excellent accuracy.
Probes may be in solution, such as in wells or on the surface of a micro-tray, or attached to a solid support. Mass modification can occur while the probes are fixed to the support, prior to fixation or upon cleavage from the support which can occur concurrently with ablation when analyzed 20 by mass spectrometry. In this regard, it can be important which strand is released from the support upon laser ablation. Preferably, in such cases, the probe is differentially attached to the support. One strand may be permanent and the other temporarily attached or, at least, selectively releasable.
Examples of solid supports which can be used include a 25 plastic, a ceramic, a metal, a resin, a gel and a membrane. Useful types of solid supports include plates, beads. microbeads, whiskers, combs, hybridization chips, membranes, single crystals, ceramics and self-assembling monolayers. A pL~fe.led embodiment comprises a two-~lim~n.~ional or three--1imen.~ional matrix, such as a gel or hybridization chipwith multiple probe binding sites (Pevzner et al., J. Biomol. Struc. & Dyn.
5 9:399-410,1991; Maskos and Southern, Nuc. Acids Res.20: 1679-84, 1992).
Hybridization chips can be used to construct very large probe arrays which are subsequently hybridized with a target nucleic acid. Analysis of the hybridization pattern of the chip can assist in the identification of the targetnucleotide sequence. Patterns can be manually or computer analyzed, but 10 it is clear that positional sequencing by hybridization lends itself to computer analysis and automation. Algorithms and software have been developed for sequence reconstruction which are applicable to the methods described herein (R. Drrnanac et al.? J. Biomol. Struc. & Dyn. 5:1085-1102, 1991; P. A. Pevzner, J. Biomol. Struc. & Dyn. 7:63-73, 1989).
Nucleic acid probes may be attached to the solid support by covalent binding such as by conjugation with a coupling agent or by, covalent or non-covalent binding such as electrostatic interactions, hydrogen bonds or antibody-antigen coupling, or by combinations thereof. Typical coupling agents include biotin/avidin, biotin/streptavidin, Staphylococcus 20 aureus protein A/IgG antibody Fc fragrnent, and streptavidin/protein A
chimeras (T. Sano and C.R. Cantor, Bio/Technology 9:1378-81, 1991), or derivatives or combinations of these agents. Nucleic acids may be attached to the solid support by a photocleavable bond, an electrostatic bond, a disul~lde bond, a peptide bond, a diester bond or a combination of these sorts 25 of bonds. The array may also be attached to the solid support by a selectively releasable bond such as 4~4'-dimethoxytrityl or its derivative.

WO 96/32504 PCTI~JS96105136 Derivatives which have been found to be useful include 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-hydroxymethyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid, and salts of these acids.
Binding may be reversible or permanent where strong ~ associations would be critical. In addition, probes may be attached to solid supports via spacer moieties between the probes of the array and the solid support. Useful spacers include a coupling agent, as described above for 10 binding to other or additional coupling partners, or to render the attachment to the solid support cleavable.
Cleavable ~t~hments may be created by attaching cleavable chemical moieties between the probes and the solid support such as an oligopeptide, oligonucleotide, oligopolyamide, oligoacrylamide, 15 oligoethylene glycerol, alkyl chains of between about 6 to 20 carbon atoms, and combinations thereof. These moieties may be cleaved with added chemical agents, electromagnetic radiation or enzymes. Examples of attachments cleavable by enzymes include peptide bonds which can be cleaved by proteases and phosphodiester bonds which can be cleaved by 20 nucleases. Chemical agents such as ~-mercaptoethanol, dithiothreitol (DTT) and other reducing agents cleave disulfide bonds. Other agents which may be useful include oxidizing agents, hydrating agents and other selectively active compounds. Electromagnetic radiation such as ultraviolet, infrared and visible light cleave photocleavable bonds. Attachments may also be 25 reversible such as, for example, using heat or enzymatic treatment, or reversible chemical or magnetic ~ chments. Release and re~ chment can be performed using, for example, magnetic or electrical fields.
Hybridized probes can provide direct or indirect information about the hybridized sequence. Direct information may be obtained from 5 the binding pattern of the array wherein probe sequences are known or can be determined. Indirect information requires additional analysis of a plurality of nucleic acids of the target array. For example, a specific nucleic acid sequence will have a unique or relatively unique molecular weight depending on its size and composition. That molecular weight can be 10 determined, for example, by chromatography (e.g HPLC), nuclear magnetic resonance (NMR), high-definition gel electrophoresis, capillary electrophoresis (e.g. HPCE), spectroscopy or mass spectrometry.
Preferably, molecular weights are determined by measuring the mass/charge ratio with mass spectrometry technology.
lS Mass spectrometry of biopolymers such as nucleic acids can be performed using a variety of techniques (e.g U.S. Patent Nos.4,442,354;
4,931,639; 5002,868; 5,130,538;5,135,870; 5,174,962). Difficulties associated with volatization of high molecular weight molecules such as DNA and RNA have been overcome, at least in part, with advances in 20 techniques, procedures and electronic design. Further, only small quantities of sample are needed for analysis, the typical sample being a mixture of 10 or so fragments. Quantities which range from between about 0.1 femtomole to about 1.0 nanomole, preferably between about 1.0 femtomole to about 1000 femtomoles and more preferably between about 10 femtomoles to 25 about 100 femtomoles are typically sufficient for analysis. These amounts can be easily placed onto the individual positions of a suitable surface or attached to a support.
Another of the important features of this invention is that it is llnn~ceSs~ly to volatize large lengths of nucleic acids to dettormine sequence S information. Using the methods of the invention, segments of the nucleic acid target, discretely isolated into separate complexes on the target array, can be sequenced and those sequence segments collated m~kin,~ it unnecçss~ry to have to volatize the entire skand at once. Techniques which can be used to volatize a nucleic acid fragment include fast atom 10 bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray, photochemical release, electrical release, droplet release, resonance ionization and combinations of these techniques.
In eleckohydrodynamic ionization, thermospray, aerospray and electrospray, the nucleic acid is dissolved in a solvent and injected with 15 the help of heat, air or electricity, directly into the ionization chamber. If the method of ionization involves a light beam, particle beam or electric discharge, the sample may be attached to a surface and inkoduced into the ionization chamber. In such situations, a plurality of samples may be attached to a single surface or multiple surfaces and introduced 20 ~imlllt~neously into the ionization chamber and still analyzed individually.
The a~pr~liate sector ofthe surface which contains the desired nucleic acid can be moved to proximate the path an ionizing beam. After the beam is pulsed on and the surface bound molecules are ionized, a different sector of the surface is moved into the path of the beam and a second sample, with the 25 same or different molecule, is analyzed without reloading the machine.
Multiple samples may also be introduced at electrically isolated regions of a surface. Different sectors of the chip are cormected to an electrical source and ionized individually. The surface to which the sample is attached may be shaped for m~x;~ efficiency ofthe ionization method used. For field ionization and field desorption, a pin or sharp edge is an efficient solid support and for particle bombardment and laser ionization, a flat surface.
The goal of ionization for mass spectroscopy is to produce a whole molecule with a charge. Preferably, a matrix-assisted laser desorption/ionization (MALDI) or electrospray (ES) mass spectroscopy is used to deterrnine molecular weight and, thus, sequence information from the target array. It will be recognized by those of ordinary skill that a variety of methods may be used which are a~ropliate for large molecules such as nucleic acids. Typically, a nucleic acid is dissolved in a solvent and injected into the ionization chamber using electrohydrodynamic ionization, thermospray, aerospray or electrospray. Nucleic acids may also be attached to a surface and ionized with a beam of particles or light. Particles which have successfully used include plasma (plasma desorption), ions (fast ion bombardment) or atoms (fast atom bombardment). Ions have also been produced with the rapid application of laser energy (laser desorption) and electrical energy (field desorption).
In mass spectrometer analysis, the sample is ionized briefly by a pulse of laser beams or by an electric field induced spray. The ions are accelerated in an electric field and sent at a high velocity into the analyzer portion of the spectrometer. The speed of the accelerated ion is directly proportional to the charge (z) and inversely proportional to the mass (m) of the ion. The mass of the molecule may be deduced from the flight characteristics of its ion. For small ions, the typical detector has a magnetic WO 96/~2504 PCT/US96/05136 field which functions to constrain the ions stream into a circular path. The radii of the paths of equally charged particles in a uniform magnetic field is directly proportional to mass. l~at is, a heavier particle with the same charge as a lighter particle will have a larger flight radius in a magnetic 5 field. It is generally considered to be impractical to measure the flight characteristics of large ions such as nucleic acids in a magnetic field because the relatively high mass to charge (m/z) ratio requires a magnet of unusual size or strength. To overcome this limitation the electrospray method, for example, can consistently place multiple ions on a molecule. Multiple 10 charges on a nucleic acid will decrease the mass to charge ratio allowing a conventional quadrupole analyzer to detect species of up to 100,000 daltons.
Nucleic acid ions generated by the matrix assisted laser desorption/ionization only have a unit charge and because of their large mass, generally require analysis by a time of flight analyzer. Time of flight 15 analyzers are basically long tubes with a detector at one end. In the operation of a TOF analyzer, a sample is ionized briefly and accelerated down the tube. After detection, the time needed for travel down the detector tube is calculated. The mass of the ion may be calculated from the time of flight. TOF analyzers do not require a magnetic field and can detect unit 20 charged ions with a mass of up to 100,000 daltons. For improved resolution, the time of flight mass spectrometer may include a reflectron, a region at the end of the flight tube which negatively accelerates ions. Moving particles emering the reflectron region, which contains a field of opposite polarity to the accelerating field, are retarded to zero speed and then reverse accelerated 25 out with the same speed but in the opposite direction. In the use of an analyzer with a reflectron, the detector is placed on the same side of the flight tube as the ion source to detect the returned ions and the effective length of the flight tube and the resolution power is effectively doubled.
The calculation of mass to charge ratio from the time of flight data takes into ac~;~t sf ~e ti~le sp~t in ~ etr~n.
S Ions with the same charge to mass ratio will typically leave the ion accelerators with a range of energies because the ionization regions of a mass spectrometer is not a point source. Ions generated further away from the flight tube, spend a longer time in the accelerator field and enter the flight tube at a higher speed. Thus ions of a single species of molecule will arrive at the detector at different times. In time of flight analysis, a longer time in the flight tube in theory provide more sensitivity, but due to the different speeds of the ions, the noise (background) will also be increased.
A reflectron, besides effectively doubling the effective length of the flight tube, can reduce the error and increase sensitivity by reducing the spread of detector impingement time of a single species of ions. An ion with a higher velocity will enter the refleckon at a higher velocity and stay in the reflectron region longer than a lower velocity ion. If the reflectron electrode voltages are arranged appropriately, the peak width contribution from the initial velocity distribution can be largely corrected for at the plane of the detector. The correction provided by the reflectron leads to increased mass resolution for all stable ions, those which do not dissociate in flight, in the spectrum.
While a linear field reflectron functions adequately to reduce noise and enhance sensitivity, reflectrons with more comple~; field strengths offer superior correctional abilities and a number of complex reflectrons can be used. The double stage reflectron has a first region with a u eaker electric WO 96/~2504 PCTIUS96105136 field and a second region with a skonger eleckic field. The quadratic and the curve field reflectron have a eleckic field which increases as a function of the distance. These functions, as their name implies, may be a quadratic or a complex exponential function. The dual stage, quadratic, and curve 5 field reflectrons, while more elaborate are also more accurate than the linear reflectron.
The detection of ions in a mass speckometer is typically performed using electron detectors. To be detected, the high mass ions produced by the mass spectrometer is converted into either electrons or low 10 mass ions at a conversion electrode. These eleckons or low mass ions are then used to start the eleckon multiplication cascade in an eleckon multiplier and further amplified with a fast linear amplifier. The signals from multiple analysis of a single sample are combined to improve the signal to noise ratio and the peak shapes, which also increase the accuracy 15 of the mass determination.
This invention is also directed to the detection of multiple primary ions directly through the use of ion cyclotron resonance and Fourier analysis. This is useful for the analysis of a complete sequencing ladder immobilized on a surface. In this method, a plurality of samples are ionized 20 at once and the ions are captured in a cell with a high magnetic field. An RF field excites the population of ions into cyclotron orbits. Because the frequencies of the orbits are a function of mass, an output signal representing the spectrum of the ion masses is obtained. This output is analyzed by a computer using Fourier analysis which reduces the combined 25 signal to its component frequencies and thus provides a measurement of the ion masses present in the ion sample. Ion cyclotron resonance and Fourier WO 96/3250~1 PCT/US96/OS136 analysis can cl~t~rmine the masses of all nucleic acids in a sample. The application of this method is especially useful on a sequencing ladder.
The data from mass spectrometry, either pe,rolllled singly or in parallel (multiplexed), can detcnnine the molecular mass of a nucleic acid S sample. The molecular mass, combined with the known sequence of the sample, can be analyzed to determine the length of the sample. Because different bases have different molecular weight, the output of a high resolution mass spectrometer, combined with the known sequence and reaction history of the sample, will determine the sequence and length of the 10 nucleic acid analyzed. In the mass spectroscopy of a sequencing ladder, generally the base sequence of the primers are known. From a known sequence of a certain length, the added base of a sequence one base longer can be clelluced by a comparison of the mass of the two molecules. This process is continlle~l until the complete sequence of a sequencing ladder is 1 5 deterrnined.
Another embodiment of the invention is directed to a method for detecting a target nucleic acid. As before, a set of nucleic acids complementary or homologous to a sequence of the target is hybridized to an array of nucleic acid probes. The molecular weights of the hybridized 20 nucleic acids determined by, for example, mass spectrometry and the nucleic acid target detected by the presence of its sequence in the sample. As the object is not to obtain extensive sequence information, probe arrays may be fairly small with the critical sequences, the sequences to be detected, repeated in as many variations as possible. Variations may have greater than 25 95% homology to the sequence of interest, greater than 80%, greater than 70% or greater than about 60%. Variations may also have additional wo 96/32s04 PC rlus96JO5136 sequences not required or present in the target sequence to increase or decrease the degree of hybridization. Sensitivity of the array to the target sequence is increased while reducing and hopefully elimin~ting the number of false positives.
Target nucleic acids to be detected may be obtained from a biological sample, an archival sarnple, an environment~l sample or another source expected to contain the target sequence. For exarnple, samples may be obtained from biopsies of a patient and the presence of the target sequence is indicative of the disease or disorder such as, for example, a neoplasm or an infection. Samples may also be obtained from environmental sources such as bodies of water, soil or waste sites to detect the presence and possibly identify or~nism~ and microor~ni.~m which may be present in the sample. The presence of particular microorg~nism.c in the sample may be indicative of a dangerous pathogen or that the normal flora is present.
Another embodiment of the invention is directed to the arrays of nucleic acid probes useful in the above-described methods and procedures. These probes comprise a first strand and a second strand wherein the first strand is hybridized to ~he second strand forming a double-stranded portion, a single-stranded portion and a variable sequence within the single-stranded portion. The array may be attached to a solid support such as a material that facilitates volatization of nucleic acids for mass spectrometry. Typically, arrays comprise large numbers vfprobes such as less than or equal to about 4R different probes and R is the length in nucleotides of the variable sequence. When utilizing arrays for large scale sequencing, larger arrays can be used whereas, arrays which are used for detection of specific sequences may be fairly small as many of the potential sequence combinations will not be necessary.
Arrays may also comprise nucleic acid probes which are e entirely single-stranded and nucleic acids which are single-stranded, but possess hairpin loops which create double-stranded regions. Such structures can function in a manner similar if not identical to the partially single-stranded probes, which comprise two strands of nucleic acid, and have the additional advantage of thermodynamic energy available in the secondary structure.
Arrays may be in solution or fixed on a solid support through streptavidin-biotin interactions or other suitable coupling agents. Arrays may also be reversibly fixed to the solid support using, for example, chemical moieties which can be cleaved with electromagnetic radiation, chemical agents and the like. The solid support may comprise materials such as matrix chemicals which assist in the volatization process for mass spectrometric analysis. Such chemicals include nicotinic acid, 3'-hydroxypicolnic acid, 2,5-dihydroxybenzoic acid, sinapinic acid, succinic acid, glycerol, urea and Tris-HCl, pH about 7.3.
Another embodiment of the invention is directed to sequencing double-stranded nucleic acids using strand-displacement polymerization. With this method it is unnecessary to denature the double-strands to obtain sequence inforrnation. Strand-displacement polymerization creates a new strand while simultaneously displacing the existing strand.
Techniques for incorporating label into the growing strand are well-know and the newly polymerized strand is easily detected by, for example, mass spectrometry.

Wo 96/32504 PcTruss6~o5l36 Target nucleic acid or nucleic acids cont~inin~ sequences that correspond to the sequence of the target are digested, for exarnple, with restriction enzymes, in one or more steps to create a set of fragments which are partially single-stranded and partially double-stranded. Another set of 5 nucleic acids, the probes, are also partially single-stranded and partially double-stranded. These probes preferably contain a variable or constant regions within the single-stranded portion of the terminus of each fragment (5'- or 3'-ovPrh~n,~.c). Probes or fragments are treated with a phosphatase LO
remove phosphate groups from the 5'-termini of the nucleic acids.
10 Phosphatase treatment prevents nucleic acid ligation by ligase which requires a t~nnin~l 5'-phosphate to covalently link to a 3'-hydroxyl. Single-stranded regions ofthe fr~gm~nt~ are hybridized to single-stranded regions of the probes forming an array of hybridized target/probe complexes.
Adjacent or abutting nucleic acid strands of the complex are ligated, 15 covalently joining a strand of the fragrnent to a strand of the probe.
Phosphatase treatment prevents both self-ligation of phosphatase-treated nucleic acids and ligation between the 5'-termini of phosphatased nucleic acids and the 3'-termini of untreated nucleic acids. These complexes are treated with a nucleic acid polymerase that recognizes and bind to the nick 20 in the unligated strand to initiate polymerization. The polymerase synthesizes a new strand using the ligated stand as a template, while displacing the complementary strand. The reaction may be supplemented with labeled or mass modified nucleotides (e.g mass modifications at positions C2, N3, N7 or C8 of purine, or at N7 or N9 of deazapurine) or 25 other detectable markers that will allow for the detection of new synthesis.
Either the probes or the fragments may be fixed to a solid support such as WO 96t32504 PCT/US96/05136 a plastic or glass surface, membrane or structure (magnetic bead) which elimin~tes the need for repetitive extractions or other purification of nucleic acids between steps.
Preferably, double-stranded nucleic acids cont~ining target 5 sequences are obtained by polymerase chain reaction or enzymatic digestion (e.g restriction enzymes) of the target sequence. Target sequences may be DNA, RNA, RNA/DNA hybrids, cDNA, PNA or modifications or combinations thereof and are preferably from about 10 to about 1,000 nucleotides in length, more preferably, from about 20 to about 500 10 nucleotides in length, and even more preferably, from about 35 to about 250 nucleotides in length. 5'-termini ofthe nucleic acid fr~gment~ orprobes may be dephosphorylated with a phosphatase, such as ~lk~line or calf intestinal phosphatase, which elimin~tes the action of a nucleic acid ligase. Upon hybridization of fragment to probe, only one of the two internal 5'-3' 15 junctions contains a 5'-phosphate and is capable of ligation. The second junction appears as a nick in a strand of the complex. Nucleic acid polymerases, such as Klenow, recognize the nick and synthesize a new strand while displacing the complementary, ligated strand. Chain elongation can proceed in the presence of, for example, nucleotide triphosphates and 20 chain termin~ting nucleotides. Nucleic acid synthesis tern~in~tes when a dideoxynucleotide is incorporated into the elongating strand. The resulting fragments represent a nested set of the sequence of the target. Precursor nucleotides may be labeled with, for example, mass modifications. The mass modified fragments can be easily analyzed by mass spectrometry to 25 determine the sequence of the target. Complexes may further comprise single-stranded binding protein (SSB; E. coli) which increases stability of wo 96/32504 PCT~S96S~5136 the complex and facilitate polymerase action. Bands otherwise obscured are more easily detecte-l SSB can be used to sequence fragments of greater than 100 nucleotides, preferably greater than 150 nucleotides and more preferably greater than 200 nucleotides.
S This method is generally useful for m~nllAl or automated nucleic acid sequencing, and especially useful for identifying and sequencing a single or group of nucleic acid species in a mixed background cont~inin~ a plurality of species of different sequences. In this method, selection is performed upon hybridization and ligation of fragments to probes. Probes may be designed to contain a cornrnon or variable sequence within the single-stranded region that is complementary to a sequence of the fragment to be identified and, if desired, sequenced. Stringency of fragment/probe hybridization can be adjusted by methods well-known to those of ordinary skill to match desired conditions of selection. For l S example, the single-stranded region of the probe can be designed to contain a specific sequence only found on the single-stranded region of the nucleic acid frA~nent of interest. ~IternAtively, multiple probes containing multiple variable regions may be used to select for those fragment sequences which may be longer than the length of the single-stranded region of any one probe. Hybridization and ligation selects the specific fragment from a complex mixture of different fragments and only that specific fragment is subsequently sequenced.
Probes are typically ~om about lS to about 200 nucleotides in length, but can be larger or small depending on the particular application.
Single-stranded regions ofthe probes may be about 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 22, 25 or 30 nucleotides in length or larger. For probes containing a variable region within the single-stranded region, the length of this variable region may be the same or smaller than the length of the entire single-stranded portion. Variable regions may be distinct between probes or common within sets of probes. The double-stranded region of the probe 5 is typically larger than the single-stranded region and may be about 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35 40 or 50 nucleotides in length or larger. Probes may also be modified to facilitate attachment to a solid support or other surfaces, or modified to be individual detectable for identification or other purposes. Sets of nucleic acids, either fragments or 10 probes, preferably contain greater than 1o2, 103, 104, 105, 1o6, 107, 1o8, 109 or 10'~ different members.
Another embodiment of the invention is directed to kits for detecting a sequence of a target nucleic acid. An array of nucleic acid probes is fixed to a solid support which may be coated with a matrix 15 chemical that facilitates volatization of nucleic acids for mass spectrometry.
Kits can be used to detect diseases and disorders in biological samples by detecting specific nucleic acid sequences which are indicative of the disorder. Probes may be labeled with detectable labels which only become detectable upon hybridization with a correctly matched target sequence.
20 Detectable labels include radioisotopes, metals, luminescent or bioluminescent chemicals, fluorescent chemicals, enzymes and combinations thereof.
Another embodiment of the invention is directed to nucleic acid sequencing systems which comprise a mass spectrometer, a computer 25 loaded with a~ op- iate software for analysis of nucleic acids and an array Wl~ ~6/;}2504 PCTJ~JS96105136 of probes which can be used to capture a target nucleic acid sequence.
Systems may be m~nll~l or automated as desired.
The following experiments are offered to illustrate embodiments of the invention, and should not be viewed as limiting the S scope of the invention.
Fx~n~les Fx~mrle 1 Preparation of Tar~et Nucleic Acid.
Target nucleic acid is prepared by restriction endonuclease cleavage of cosmid DNA. The properties of type II and other restriction 10 nucleases ~at cleave outside of their recognition sequences were exploited.
A restriction digestion of a 10 to 50 kb DNA sample with such an enzyme produced a mixture of DNA fragments most of which have unique ends.
Recognition and cleavage sites of useful enzymes are shown in Table 1.
Table 1 Restriction Enzymes and Recognition Sites for PSBH
Mwo I GCNNNNN-NNGC
CGNN-NNNNNCG

~si YI CCNNNNN-NNGG
GGNN-NNNNNCC
t Apa BI GCANNNNN-TGC
CGT-NNNNNACG
t Mnl I CCTCN7 t WO 96/32504 PCI~/US96/OS136 Tsp RI NNCAGTGNN
NNGTCACNN

S Cje I CCANNNN~N-GFNNNN
GG~NN~N-CANNNN

Cje PI CCANM~N-NNTCNN
1 0 GG~NNNN-NNAGNN

One restriction enzyme, ~paB 15, with a 6 base pair recognition site may also be used. DNA sequencing is best served by 15 enzymes that produce average fragment lengths comparable to the lengths of DNA sequencing ladders analyzable by mass speckometry. At present these lengths are about 100 bases or less.
BsiYI and Mwo I restriction endonucleases are used together to digest DNA in preparation of PSBH. Target DNA from is cleaved to 20 completion and complexed with PSBH probes either before or after melting.
The fraction of fragments with unique ends or degenerate ends depends on the complexity of the target sequence. For example, a 10 kilobase clone would yield on average 16 fr~f~nent~ or a total of 32 ends since each double-stranded DNA target produces two ligatable 3' ends. With 1024 possible 25 ends, Poisson statistics (Table 2) predict that there would be 3%
degeneracies. In contrast, a 40 kilobase cosmid insert would yield 64 fragments or 128 ends, of which, 12% of these would be degenerate and a 50 kilobase sample would yield 80 fragments or 160 ends. Some of these would surely be degenerate. Up to at least 100 kilobase, the larger the target 30 the more sequence are available from each multiple~ DNA sample preparation. With a 100 kilobase t~rget, 27% of the targets would be degenerate.
Table 2 Poisson Distribution of Restriction Enzyme Sites Targetsize Mwo I TspR I
(kb) Sequencing Assembly Sequencing Assembly 0.97 0.60 0.94 0.94 0.88 0.14 0.80 0.80 100 0.73 0.01 0.57 0.57 With BsiYI and Mwo I, any restriction site that yields a unique 5 base end may be captured twice and the resulting sequence data obtained will read away from the site in both directions (Figure 5). With the knowledge of three bases of overlapping sequence at the site, this sorts all 15 sequences into 64 different categories. With 10 kilobase targets, 60% will contain fragments and, thus sequence assembly is automatic.
Two array capture methods can be used with Mwo I and BsiY
I. In the first method, conventional five base capture is used. Because the two target bases adjacent to the capture site are known, they from the restriction enzyme recognition sequence, an alternative capture strategy would build the complement of these two bases into the capture sequence.
Seven base capture is thermodynamically more stable, but less discrimins~ting against mi~m~tches.
TspR I is another commercially available restriction enzyme with properties that are very attractive for use in PSBH-mediated Sanger sequencing. The method for using TspR I is shown in Figure 6. TspR I has a five base recognition site and cuts two bases outside this site on each strand to yield nine base 3' single-stranded overhangs. These can be captured with partially duplex probes with complementary nine base WO 96/32504 PCI~/US96/05136 overh~n~c. Because only four bases are not specified by enzyme recognition, TspR I digest results in only 256 types of cleavage sites. With human DNA the average fr~gment length that results is 1370 bases. This enzyme is ideal to generate long Sequence ladders and are useful to input to 5 long thin gel sequencing where reads up to a kilobase are common. A
typical human cosmid yields about 30 ~spR I fragments or 60 ends. Given the length distribution expected, many of these could not be sequenced fully from one end. With 256 possible overhangs, Poisson statistics (Table 2) indicate that 80% adjacent fragments can be assembled with no additional 10 labor. Thus, very long blocks of continuous DNA sequence are produced.
Three additional restriction enzymes are also useful. These are Mnl I, Cje I and CjeP I (Table 1). The first has a four base site with one A+T should give smaller human DNA fragments on average than Mwo I or BsiY I. The latter two have unusual interrupted five base recognition sites 15 and might supplement TspR I.
Target DNA may also be prepared by tagged PCR. It is possible to add a preselected five base 3' terminal sequence to a target DNA
using a PCR primer five bases longer than the known target sequence priming site. Samples made in this way can be captured and sequenced 20 using the PSBH approach based on the five base tag. A biotin was used to allow purification of the complementary strand prior to use as an immobilized sequencing template. A biotin may also be placed on the tag.
After capture of the duplex PCR product by streptavidin-coated magnetic microbeads, the desired strand (needed to serve as a sequencing template) 25 could be flçn~tl~red from the duplex and used to contact the entire probe array. For multiplex sample preparation, a series of different five base WO 96/~2504 PCT~S96105136 tagged primers would be employed, ideally in a single multiplex PCR
reaction This approach also requires knowing enough target sequence for unique PCR amplification and is more useful for shotgun sequencing or co~ e sequencing than for de novo sequencing.
5 Fx~mrle 2 R~ic Aspects of Positional Sequenc;r~ by Hybridization.
An ex~min~tion of the potential advantages of stacking hybridization has been carried out by both calculations and pilot experim~nt~ Some c~lc~ te(l Tm's for perfect and mi~m~tçhed duplexes are shown in Figure 7. These are based on average base compositions. The 10 calculations revealed that the binding of a second oligomer next to a pre-formed duplex provides an extra stability equal to about two base pairs and that mis-pairing seems to have a larger consequence on stacking hybridization than it does on ordinary hybridization. Other types of mis-pairing are less destabilizing, but these can be elimin~te~l by requiring a l5 ligation step. In standard SBH, a terminal mi~m~tch is the least destabilizing event, and leads to the greatest source of ambiguity or background. For an octanucleotide complex, an average terminal mi~m~tch leads to a 6~C lowering in Tm. For stacking hybridization, a terminal mi~m~tch on the side away from the pre-existing duplex, is the least 20 destabilizing event. For a pentamer, this leads to a drop in Tm of 10~C.
These considerations indicate that the discrimin~tion power of stacking hybridization in favor of perfect duplexes are greater than ordinary SBH.
Example 3 Preparation of Model Arrays.
In a single synthesis, all 1024 possible single-stranded probes 25 with a constant 18 base stalk followed by a variable 5 base extension can be created. The 18 base extension is designed to contain two restriction WO 96/32~i04 PCT/US96/05136 enzyme cutting sites. Hga I generates a S base, 5' overhan~, consisting of the variable bases N5. Not I generates a 4 base, 5' overhang at the constant end of the oligonucleotide. The synthetic 23-mer mixture hybridized with a complementary 1 8-mer forms a duplex which can be enzymatically 5 extended to form all 1024, 23-mer duplexes. These are cloned by, for example, blunt end ligation, into a plasmid which lacks Not I sites. Colonies containing the cloned 23-base insert are selected and each clone contains one unique sequence. DNA mi~ reL)s can be cut at the constant end of the stalk, filled in with biotinylated pyrimidines and cut at the variable end of 10 the stalk to generate the 5 base 5' overhang. The resulting nucleic acid is fractionated by Qiagen columns (nucleic acid purification columns) to discard the high molecular weight material. The nucleic acid probe will then be attached to a ~ avidin-coated surface. This procedure could easily be automated in a Beckman Biomec or equivalent chemical robot to produce l S many identical arrays of probes.
The initial array contains about a thousand probes. The particular sequence at any location in the array will not be known.
However, the array can be used for statistical evaluation of the signal to noise ratio and the sequence discrimination for different target molecules 20 under different hybridization conditions. Hybridization with known nucleic acid sequences allows for the identification of particular elements of the array. A sufficient set of hybridizations would train the array for any subsequent sequencing task. Arrays are partially characterized until they have the desired properties. For example, the length of the oligonucleotide 25 duplex, the mode of its ~ chment to a surface and the hybridization conditions used can all be varied using the initial set of cloned DNA probes.

WO 96/32504 PCTIUS96~05136 Once the sort of array that works best is determined, a complete and fully characterized array can be constructed by ordinary chemical synthesis.
Example 4 P~ al~lion of Specific Probe Arrays.
With positional SBH, one potential trick to compensate for 5 some variations in stability among species due to GC content variation is to provide GC rich st~king duplex adjacent AT rich overhangs and AT rich stacking duplex adjacent GC rich overh~n~s. Moderately dense arrays can be made using a typical x-y robot to spot the biotinylated comp~unds individually onto a ~ t~vidin-coated surface. Using such robots, it is 10 possible to make arrays of 2 x 104 samples in 100 to 400 cm2 of nominal surface. Commercially available ~ avidin-coated beads can be adhered, permanently to plastics like polystyrene, by exposing the plastic first to a brief treatment with an organic solvent like triethylamine. The resulting plastic surfaces have enormously high biotin binding capacity because of the 15 very high surface area that results.
In certain experiments, the need for attaching oligonucleotides to surfaces may be circumvented altogether, and oligonucleotides attached to streptavidin-coated magnetic microbeads used as already done in pilot experiments. The beads can be manipulated in microtiter plates. A
20 magnetic separator suitable for such plates can be used including the newly available compressed plates. For example, the 18 by 24 well plates (Genetix, Ltd.; USA Scientific Plastics) would allow cont~inment of the entire arrav in 3 plates. This format is well handled by existing chemical robots. It is preferable to use the more compressed 36 by 48 well format so 25 the entire array would fit on a single plate. The advantages of this approachfor all the experiments are that any potential complexities from surface effects can be avoided and already-existing liquid h~n~lling, therm~l control and imaging methods can be used for all the experiments.
Lastly, a rapid and highly efficient method to print arrays has been developed. Master arrays are made which direct the preparation of S replicas or a~)pr~rlate complementary arrays. A master array is made manually (or by a very accurate robot) by sampling a set of custom DNA
sequences in the desired pattern and then transferring these sequences to the replica. The master array is just a set of all 1024-4096 compounds printed by multiple he~e~l pipettes and compressed by offsetting. A potentially 10 more elegant approach is shown in Figure 8. A master array is made and used to transfer components of the replicas in a sequence-specific way. The sequences to be transferred are designed to contain the desired 5 or 6 base 5' variable overhang adjacent to a unique 15 base DNA sequence.
The master array consists of a set of streptavidin bead-15 impregnated plastic coated metal pins. Immobilized biotinylated DNAstrands that consist of the variable 5 or 6 base segment plus the constant 15 base segment are at each tip. Any unoccupied sites on this surface are filled with excess free biotin. To produce a replica chip, the master array is incubated with the complement of the 15 base constant sequence, 5'-labeled 20 with biotin. Next, DNA polymerase is used to synthesize the complement of the 5 or 6 base variable sequence. Then the wet pin array is touched to the streptavidin-coated surface ofthe replica and held at a temperature above the Tm of the complexes on the master array. If there is insufficient liquid carryover from the pin array for efficient sample transfer, the replica array 25 could first be coated with spaced droplets of solvent, either held in concavecavities or delivered by a multi-head pipettor. After the transfer, the replica WO 96/32504 PC~IUS96J053.36 chip is incllb~tecl with the complement of 15 base con~t~nt sequence to reform the double-stranded portions of the array. The basic advantage of this scheme is that the master array and transfer compounds are made only once aIld the m~nllf~chlre of replica arrays can proceed almost endlessly.
5 Example 5 AttachmeIlt ofNucleic Acids Probes to Solid Supports.
Nucleic acids may be attached to silicon wafers or to beads.
A silicone solid support was d~.iv~ ed to provide iodoacetyl fimctionalities on its surface. Derivatized solid support were bound to disulfide containing oligodeoxynucleotides. ~ltern~tively~ the solid support may be coated with 10 ~llc~idin or avidin arld bound to biotinylated DNA.
Covalent ~ chment of oligonucleotide to derivatized chips:
Silicon wafers are chips with an approximate wei~ht of 50 mg To m~int~in uniform reaction condition, it was necessary to determine the exact weight of each chip and select chips of similar weights for each experiment. The 15 reaction scheme for this procedure is shown in Figure 9.
To derivatize the chip to contain the iodoacetyl functionality an anhydrous solution of 25% (by volume) 3-aminopropyltrieshoxysilane in toluene was prepared under argon and aliquotted (700 111) into tubes. A
50 mg chip requires approximately 700 ~11 of silane solution. Each chip was 20 flamed to remove any surface cont~rnin~nts during its manufacture and dropped into the silane solution. The tube containing the chip was placed under an argon environment and shaken for approximately three hours.
~fter this time, the silane solution was removed and the chips were washed three times with toluene and three times with dimethyl sulfoxide (DMSO).
25 A 10 mM solution of N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) (Pierce Chemical Co.; Rockford, IL) was prepared in anhydrous DMSO and added to the tube cont~inin~ a chip. Tubes were shaken under an argon environment for 20 minutes. The SIAB solution was removed and after three washes with DMSO, the chip was ready for attachment to oligonucleotides.
Some oligonucleotides were labeled so the efficiency of ~ çhment could monitored. Both 5' disulfide cont~ining oligodeoxynucleotides and unmodified oligodeoxynucleotides were radiolabeled using terminal deoxynucleotidyl transferase enzyme and standard techniques. In a typical reaction, 0.5 mM of disulfide-containing oligodeoxynucleotide mix was added to a trace amount of the same species that had been radiolabeled as described above. This mixture was incubated with dithiothreitol (DTT) (6.2 ,umol, 100 mM) and ethylenediaminetetraacetic acid (EDTA) pH 8.0 (3 ,umol, 50 mM). EDTA
served to chelate any cobalt that remained from the radiolabeling reaction that would complicate the cleavage reaction. The reaction was allowed to proceed for 5 hours at 37~C. With the cleavage reaction essentially complete, the free thiol-containing oligodeoxynucleotide was isolated using a Chromaspin- 10 column.
Similarly, Tris-(2-carboxyethyl)phosphine (TCEP) (Pierce Chemical Co.; Rockford, IL) has been used to cleave the disulfide.
Conditions utilize TCEP at a concentration of approximately 100 mM in pH
4.5 buffer. It is not necessary to isolate the product following the reaction since TCEP does not competitively react with the iodoacetyl functionality.
To each chip which had been derivatized to contain the iodoacetyl functionality was added to a 10 ,uM solution of the oligodeoxynucleotide at pH 8. The reaction was allo-~ed to proceed WO 96/32504 PCTIUS96JOS1~6 overnight at room temperature. In ~is manner, two different oligodeoxynucleotides have been t;x~llilled for their ability to bind to the iodoacetyl silicon wafer. T~e first was the free thiol cont~inin~
oligodeoxynucleotide already described. In parallel with the free thiol 5 cont~ining oligodeoxynucleotide reaction, a negative control reaction has been performed that employs a S' unmodified oligodeoxynucleotide. This species has similarly been 3' radiolabeled, but due to the unmodified 5' terrninus, the non-covalent, non-specific interactions may be determined.
Following the reaction, the radiolabeled oligodeoxynucleotides were 10 removed and the chips were washed 3 times with water and quantitation proceeded.
To ~letçrrnine the efficiency of ~tt~c~ment~ chips of the wafer were exposed to a phosphorimager screen (Molecular Dynamics). This exposure usually proceeded overnight, but occasionally for longer periods 15 of time depending on the amount of radioactivity incorporated. For each different oligodeoxynucleotide l.tili7~-1 reference spots were made on polystyrene in which the molar amount of oligodeoxynucleotide was known.
These reference spots were also exposed to the phosphorimager screen.
Upon sc~nning the screen, the quantity (in moles) of oligodeoxynucleotide 20 bound to each chip was determined by c~ g the counts to the specific activities of the references. Using the weight of each chip, it is possible to calculate the area of the chip:
(g of chip) ( 1 130 mm2/g) = x mm2 By incorporating this value, the amount of oligodeoxynucleotide bound to 25 each chip mav be reported in fmol/mm2. It is necessary to divide this value by two since a radioactive signal of 32p iS strong enough to be read through the silicon wafer. Thus the instrument is essentially recording the radioactivity from both sides of the chip.
Following the initial qll~ntit~tion each chip was washed in 5 x SSC buffer (75 rnM sodium citrate, 750 rnM sodium chloride, pH 7) with 5 50% formamide at 65~C for 5 hours. Each chip was washed three times with warm water, the S x SSC wash was repeated, and the chips requantitated. Disulfide linked oligonucleotides were removed from the chip by incubation with 100 rnM DTT at 37~C for 5 hours.
Example 6 Attachment of Nucleic Acids to Streptavidin Coated Solid 10 Support.
Immobilized single-stranded DNA targets for solid-phase DNA sequencing were prepared by PCR amplification. PCR was performed on a Perkin Elmer Cetus DNA Thermal Cycler using VentR (exo~) DNA
polymerase (New England Biolabs; Beverly, MA), and dNTP solutions 15 (Promega; Madison, WI). EcoR I digested plasmid NB34 (a PCRTM II
plasmid with a one kb target anonymous human DNA insert) was used as the DNA template for amplification. PCR was performed with an 1~-nucleotide upstream primer and a downstream 5'-end biotinylated 18-nucleotide primer. PCR amplification was carried out in a 100 ~11 or 400 ~11 20 volume containing 10 mM KCI, 20 mM Tris-HCI (pH 8.8 at 25~C), 10 mM
(NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 250 ~lM dNTPs, 2.5 ,uM
biotinylated primer, 5 ~lM non-biotinylated primer, less than 100 ng of plasmid DNA, and 6 units of Vent (exo~) DNA polymerase per 100 ~1 of reaction volume. Thirty temperature cycles were performed which included 25 a heat denaturation step at 9~~C for 1 minute, followed by annealing of primers to the template DNA for 1 minute at 60~C, and DNA chain wo 96/32504 PC~IUS96105136 extension with Vent (exo~) polymerase for 1 minute at 72~C. For amplification with the tagged primer, 45~C was selected for primer ~nne~lin~ The PCR product was purified through a Ultrafree-MC 30,000 NMVVL filter unit (Millipore, Bedford, MA) or by electrophoresis and S extraction from a low melting agarose gel. About 10 pmol of purified PCR
fragment was mixed with 1 mg of prewashed magnetic beads coated with fidin (Dynabeads M280, Dynal, Norway) in 100 ~1 of 1 M NaCl and TE incubating at 37~C or 45~C for 30 minutes.
The magnetic beads were used directly for double stranded 10 sequencing. For single stranded sequencing, the immobilized biotinylated double-stranded DNA fragment was converted to single-stranded form by treating with freshly prepared 0.1 M NaOH at room temye~aLllFe fsr 5 minl-tes. The magnetic beads, with immobilized single-stranded DNA, were washed with 0.1 M NaOH and TE before use.
15 Example 7 Hybri(1i7~tion Specificity.
Hybridization was performed using probes with five and six base pair overhangs, including a five base pair match, a five base pair mi~m~tch, a six base pair match, and a six base pair mismatch. These sequences are depicted in Table 3.

Table 3 Hybridized Test Sequences Test Sc~ s.
5 bp overlap, perfect match:

3'-TCG AGA ACC TTG GCT*-S' (SEQ ID NO I ) 3'-CTA CTA GGC TGC GTA GTC(SEQ ID NO 2) S'-biotin-GAT GAT CCG ACG CAT CAG AGC TC-3' (SEQ ID NO 3) 5 bp overlap, ,..;~.,.;1l. 1) at 3' end:
3'-TCG AGA ACC TTG GCT*-5' (SEQ ID NO 1) 3'-CTA CTA GGC TGC GTA GTC(SEQ ID NO 2) 5'-biotin-GAT GAT CCG ACG CAT CAG AGC 1~-3' (SEQ ID NO 4) 6 bp overlap, perfect match:
3'-TCG AGA ACC TTG GCT*-5' (SEQ ID NO I ) 3'-CTA CTA GGC TGC GTA GTC(SEQ ID NO 2) 15 5'-biotin-GAT GAT CCG ACG CAT CAG AGC TCT-3' (SEQ ID NO 5) 6 bp overlap, ~..;~ four bases ~om 3' end:
3'-TCG AGA ACC TTG GCT*-5' (SEQ ID NO 1) 3'-CTA CTA GGC TGC GTA GTC(SEQ ID NO 2) 5'-biotin-GAT GAT CCG ACG CAT CAG AGT TCT-3' (SEQ ID NO 6) The biotinylated double-stranded probe was prepared in TE
buffer by annealing the complimentary single strands together at 68~C for five minutes followed by slow cooling to room tempcla~u,e. A five-fold 25 excess of monodisperse, polystyrene-coated magnetic beads (Dynal) coated with streptavidin was added to the double-stranded probe, which as then incubated with agitation at room temperature for 30 minlltes. After ligation, the samples were subjected to two cold (4~C) washes followed by one hot (90~C) wash in TE buffer (Figure 10). The ratio of 32p in the hot 30 supern~t~nt to the total amount of 32p was determined (Figure 11). At high NaCl concentrations~ mi.cms~tched target sequences were either not annealed or were removed in the cold washes. Under the same conditions, the matched target sequences were annealed and ligated to the probe. The final hot wash removed the non-biotinylated probe oli~onucleotide. This WO 96t32504 PCTIUS961~)5136 oligonucleotide contained the labeled target if the target had been ligated to ~e probe.
Fx~mple 8 Con~pens~tin~ for Variation~ in Base Con~l?osition.
The Dependence on TM on base composition, and on base 5 sequence may be overcome with ~e use of salts like te~amethyl ammonium halides or betaines. ~It~rn~tively, base analogs like 2,6-diamino purine and 5-bromo U can be used instead of A and T, respectively, to increase the stability of A-T base pairs, and derivatives like 7-deazaC~ can be us;,d to decrease the stability of G-C base pairs. The initial Experiments shown in 10 Table 2 indicate that the use of enzymes will elimin~te many of the complications due to base sequences. This gives the approach a very significant advantage over non-enzymatic methods which require different conditions for each nucleic acid and are highly matched to GC content.
Another approach to compensate for differences in stability is 15 to vary the base next to the stacking site. Experiments were performed to test the relative effects of all four bases in this position on overall hybridization discrimin~tion and also on relative ligation discrimination other base analogs such as dU (deoxyuridine) and 7-deazaG may also be useful to suppress effects of secondary structure.
20 Example 9 nNA T ig~tion to Oligonuçleotide Arrays.
E. coli and T4 DNA ligases can be used to covalently attach hybridized target nucleic acid to the correct immobilized oligonucleotide probe. This is a highly accurate and efficient process. Because ligase absolutel~ requires a correctly base paired 3' terminus, ligase will read only 25 the 3'-terrninal sequence of the target nucleic acid. After ligation, the resulting duplex will be 23 base pairs long and it will be possible to remove unhybridized, unligated target nucleic acid using fairly stringent washing conditions. A~r~ol)l;ately chosen positive and negative controls n~1rate the specificity of ~is method, such as arrays which are lacking a 5'-tçrrnin~l phosphate adjacent to the 3' overhang since these probes will 5 not ligate to the target nucleic acid.
There are a number of advantages to a ligation step. Physical specificity~ is supplanted by enzymatic specificity. Focusing on the 3' end ofthe target nucleic also minimi7e problems arising from stable secondary structures in the target DNA. DNA ligases are also used to covalently attach 10 hybridized target DNA to the correct immobilized oligonucleotide probe.
Several tests of the feasibility of the ligation method shown in Figure 12.
Biotinylated probes were attached at 5' ends (Figure 12A) or 3' ends (Figure 12B) to ~,L~c~lavidin-coated magnetic microbeads, and annealed with a shorter, complementary, constant sequence to produce duplexes with 5 or 15 6 base single-stranded overhangs. 32P-end labeled targets were allowed to hybridize to the probes. Free targets were removed by capturing the beads with a magnetic separator. DNA ligase was added and ligation was allo~ ed to proceed at various salt concentrations. The samples were washed at room temperature, again manipulating the immobilized compounds with a 20 magnetic separator to remove non-ligated material. Finally, samples w ere incubated at a temperature above the Tm of the duplexes, and eluted single strand was retained after the remainder of the samples were removed b~
mag~netic separation. The eluate at this point consisted of the li~~ated material. The fraction of ligation was estimated as the amount of 3'P
25 recovered in the high temperature wash versus the amount recovered in both the hi ~h and low temperature washes. Results indicated that salt conditions -WO 96/;~2504 PCTIUS96105136 caIl be found where the ligation proceeds efficiently with perfectly matched 5 or 6 base ov~rh~n~, but not wi~ G-T mi~m~tçl~es. The results of a more extensive set of similar experiments are shown in Tables 4-6.
Table 4 looks at the effect of the position of the mi~m~t~h and 5 Table S examines the ef~ect of base composition on the relative discrimination of perfect matches verses weakly destabilizing mi.~m~tçlles.
These data demonstrate that effective discrimin~tion between perfect matches and single mi~m~t~l~es occurs with all five base overhangs tested and that there is little if any effect of base composition on the amount of 10 ligation seen or the effectiveness of match/mi~m~tch discrimin~tion. Thus, the serious problems of ~ lin~ with base composition effects on stability seen in ordinary SBH do not appear to be a problem for positional SBH.
Furthermore, as the worst micm~teh position was the one distal ~om the phosphodiester bond formed in the ligation reaction, any mi~m~tçhes that 15 survived in this position would be elimin~ted by a polymerase extension reaction. A polymerase such as Sequenase version 2, that has no 3'-endonuclease activity or terminal transferase activi~y would be usefill in this regard. Gel electrophoresis analysis confirrned that the putative ligation products seen in these tests were indeed the actual products synthesized.
Table 4 Ligation Efficiency of Matched and Mismatched Duplexes in 0.2 M NaCI at 37~C
(SEQ ID NO 1) 3'-TCC AGA ACC TTG GCT-S' Lip:~tion Efficicncy - CTA CTA GGC TGC GTA GTC-S'(SEQ ID NO 2) S'-B- GAT GAT CCG ACG CAT CAG AGC TC 0.170 (SEQ ID NO 3) S'-B- GAT GAT CCG ACG CAT CAG AGC TT 0.006 (SEQ ID NO 4) S'-B- GAT GAT CCG ACG CAT CAG AGC TA 0.006 (SEQ ID NO 7) 30 S'-B- GAT GAT CCG ACG CAT CAG AGC CC 0.002 (SEQ ID NO 8) S'-B- GAT GAT CCG ACG CAT CAG AGT TC 0.004 (SEQ ID NO 9) WO 96/32~i04 PCT/US96/05136 S'-B-GAT GAT CCG ACG CAT CAG AAC TC 0.001 (SEQ ID NO 10) Table S
Ligation Efficiency of Matched and Mismatched Duplexes in 0.2 M NaCI at 37~C and its Dependance on AT Content of the Overhang Overh~ng Sequences ~T Content Li~tionFfficiency Match GGCCC 0/5 0.30 Mi.cm~tch GGCCT 0.03 Match AGCCC 1/5 0.36 15 Mi.~m~tch AGCTC 0-.02 Match AGCTC 2/5 0.17 Mi~m~tch AGCTT 0.01 Match AGATC 3/5 0.24 Mi~m~tch AGATT 0.01 Match ATATC 4/5 0.17 Mi~m~tch ATATT 0.01 Match ATATT 5/5 0.31 Micm~tch ATATC Q.02 wo 96/32504 PC rnJSs6~05136 Table 6 a Increasing Di~.in~tion by Sequencing Extension at 37~C
T.~p~tion F.fficjency niP~fion l~xtension (c~m) S (percent) (+) (-) (SEQID NO 1) 3'-TCG AGA ACC TTG GCT-5'*
CTA CTA GGC TGC GTA GTC-5'(SEQID NO 2) 5'-B- GAT GAT CCG ACG CAT CAG AGA TC 0.24 4,934 29,500 0 (SEQIDNO 11) 5'-B- GAT GAT CCG ACG CAT CAG AGC TT 0.01 116 ~Q
(SEQID NO 4) Dis.;.;.. ;.. ~;cn= x24 x42 x118 (SEQID NO 1) 3'-TCG AGA ACC TTG GCT-5'*
CTA CTA GGC TGC GTA GTC-5'(SEQID NO 2) 5'-B- GAT GAT CCG ACG CAT CAG ATA TC 0.17 12,250 25,200 (SEQID NO 12) 5'-B- GAT GAT CCG ACG CAT CAG ATA TT 0.01 ~Q ~Q
(SEQID NO 13) Di.s~ n = x17 x51 x65 "B"--Biotin The discrimination for the correct sequence is not as great with an external mi.~m~tch (which would be the most difficult case to discrimin~te) as with an internal mismatch (Table 6). A mismatch right at the ligation point would presumably offer the highest possible 30 discrimination. In any event, the results shown are very promising. Already there is a level of discrimin~tion with only 5 or 6 bases of overlap that is better than the discrimin~tion seen in conventional SBH with 8 base overlaps.
Example 10 Capture and Sequencin~ of a Tar~et Nucleic Acid.
A mixture oftarget DNA was prepared by mixing equal molar ratio of eight different oligos. For each sequencing reaction, one specific - partially duple~; probe and eight different targets were used. The sequence of the probe and the targets are shown in Tables 7 and 8.

Table 7 Duplex Probes Used (DF25) 5'-F-GATGATCCGACGCATCAGCTGTG (SEQID NO 14) 53'-CTACTAGGCTGCGTAGTC (SEQID NO 2) (DF37) 5'-F-GATGATCCGACGCATCACTCAAC(SEQID NO 15) 3'-CTACTAGGCTGCGTAGTG (SEQID NO 2) 0(DF22) 5'-F-GATGATCCGACGCATCAGAATGT(SEQIDNO 16) 3'-CTACTAGGCTGCGTAGTC (SEQID NO 2) (DF28) 5'-F-GATGATCCGACGCATCAGCCTAG(SEQID NO 17) 53'-CTACTAGGCTGCGTAGTC (SEQID NO 2) (DF36) S'-F-GATGATCCGACGCATCAGTCGAC(SEQID NO 18) 3'-CTACTAGGCTGCGTAGTC (SEQID NO 2) (DFlla)5'-F-GATGATCCGACGCATCACAGCTC(SEQID NO 19) 203'-CTACTAGGCTGCGTAGTG (SEQID NO 2) (DF8a) 5'-F-GATGATCCGACGCATCAAGGCCC(SEQID NO 20) 3'-CTACTAGGCTGCGTAGTT (SEQID NO 2) 25Table 8 Mixture of Targets (NB4) 3'-TTACACCGGATCGAGCCGGGTCGATCTAG (DF22) (SEQID NO 1) (NB4-5) 3'-GGATCGACCGGGTCGATCTAG (DF28) (SEQID NO '') (DF5) 3'-AGCTGCCGGATCGAGCCGGGTCGATCTAG (DF36) (SEQID NO ~3) (TSI0) 3'-TCGAGAACCTTGGCT (DFlla) (SEQID NO 24) 35 (NB3.10) 3'CCGGGTCGATCTAG (DF8a) (SEQID NO ~5) Micm~trh (NB3.4) 3'-CCGGATCAAGCCGGGTCGATCTAG(DF8a) (SEQID NO ~6) (NB3.7) 3'-TCAAGCCGGGTCGATCTAG (DFlla) (SEQID NO ~7) 40 (NB3-9) 3'-AGCCGGGTCGATCTAG (DF36)(SEQID NO '8) Two pmol of each of the two duplex-probe-forming oligonucleotides and 1.5 pmol of each of the eight different targets were 45 mixed in a 10 ~11 volume containing 2 ,ul of Sequenase buffer stock (200 mM

Tris-HCl, pH 7.5, 100 mM MgC12, and 250 mM NaCl) from the Sequenase kit. The ~nn~lin~ mixture was heated to 65 ~C and allowed to cool slowly to room tt~ ~d~ . While the reaction mixture was kept on ice, 1 ~11 0.1 M dithiothreitol solution, 1 ~Ll Mn buf~er (0.15 M sodium isocitrate and 0.1 5 M MnCl2), and 2 ~1 of diluted Sequenase (1.5 units) were mixed, and the 2 ~11 of reaction mixture was added to each of the four teImination mixes at room temperature (each consisting of 3 ~1 of the a~l~liate termination mix: 16 ~lM dATP, 16 IlM dCTP, 16 ,uM dGTP, 16 ~LM dTTP and 3.2 ~M
of one of the four ddNTPs, in 50 mM NaCl). The reaction mixtures were 10 fur~er incubated at room temperature for 5 minl~tes, and termin~t~rl with the addition of 4 ~11 of Ph~rm~cia stop mix (deionized formamide cont~ining dextran blue 6 mg/ml). Samples were denatured at 90-95 ~C for 3 minl~tes and stored on ice prior to loading. Sequencing samples were analyzed on an ALF DNA sequencer (Pharmacia Biotech; Piscataway, NJ) using a 10%
15 polyacrylamide gel containing 7 M urea and 0.6 x TBE. Sequencing results from the gel reader are shown in Figure 13 and summarized in Table 9.
Matched targets hybridized correctly and are sequenced, whereas mism~tched targets do not hybridize and are not sequenced.

Table 9 Sllmm~ry of Hybridization Data Reaction ~Iybridization Sequence Comment S 1 Probe: DF25 Target: mixture No mi~ tch 2 Probe: DF37 Target: mixture No micm~tch 3 Probe: DF22 Target: mixture Yes match 4 Probe: DF28 Target: mixture Yes match Probe: DF36 Target: mixture Yes match 6 Probe: DFl la Target: mixture Yes match 7 Probe: DF8a Target: mixture Yes match 8 Probe: DF8a Target: NB3.4 No mismatch 9 Probe: DF8a Target: TS12 No mi~m~tch Probe: DF37 Target: DF5 No mismatch Example 11 Elon~tion of Nucleic Acids Bound to Solid Su~orts.
Elongation was carried out either by using Sequenase version 2.0 kit or an AutoRead sequencing kit (Ph~ cia Biotech; Piscataway, NJ) employing T7 DNA polymerase. Elongation of the immobilized single-20 stranded DNA target was performed with reagents from the sequencing kitsfor Sequenase Version 2.0 or T7 DNA polymerase. A duplex DNA probe containing a 5-base 3' overhang was used as a primer. The duplex has a 5'-fluorescein labeled 23-mer, containing an 1 8-base 5' constant region and a 5-base 3' variable region (which has the same sequence as the 5'-end of the 25 corresponding nonbiotinylated primer for PCR amplification of target DNA, and an 1 8-mer complementary to the constant region of the 23-mer. The duplex was formed by annealing 20 pmol of each of the two oligonucleotides in a 10 ,ul volume containing 2 ~11 of Sequenase buffer stock (200 mM Tris-HCI, pH 7.5, 100 mM MgCI2, and 250 mM NaCI) from 30 the Sequenase kit or in a 13 ,ul volume containing 2 1ll of the annealing buffer ( 1 M Tris-HCI, pH 7.6, 100 mM MgCI2) from the AutoRead WO g6/32504 PCT/USS~610SI36 sequencing kit. The ~nne~ling mixture was heated to 65 ~C and allowed to cool slowly to 37~C over a 20-30 minllte time period. The duplex primer was ~nnto~led with the immobilized single-stranded DNA target by adding the ~nne~lin~ mixture to the DNA-cont~ining magnetic beads and the 5 resulting mixture was further incubated at 37~C for 5 minlltes, room temperature for 10 minlltes, and finally 0~C for at least 5 minlltes. For Sequenase reactions, 1 ,ul 0.1 M dithiothreitol solution, 1 ~11 Mn buffer (~.15 M sodium isocitrate and 0.1 M MnCl2) for the relative short target, and 2 ~11 of diluted Sequenase (1.5 units) were added, and the reaction mixture was 10 divided into four ice cold termination mixes (each consists of 3 ~11 of the al.~r~liate termin~tion mix: 80 ~lM dATP, 80 ~lM dCTP, 80 ,uM dGTP, 80 ~lM dTTP and 8 ~lM of one of the four ddNTPs, in 50 mM NaCl). For T7 DNA polymerase reactions, 1 ~1 of extension buffer (40 mM McCk, pH 7.5, 304 mM citric acid and 324 mM DTT) and 1 ,ul of T7 DNA polymerase (8 15 units) were mixed, and the reaction volume was split into four ice cold termination mixes (each consisting of 1 ,ul DMSO and 3 ~11 of the appropriate termination mix: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP and 5 IlM of one of the four ddNTPs, in 50 mM NaCI and 40 mM
Tris-HCl, pH 7.4). The reaction mixtures for both enzymes were further 20 incubated at 0~C for 5 minutes, room temperature for 5 minutes and 37~C
for 5 minutes. After the completion of extension, the supernatant was removed. and the magnetic beads were re-suspended in 10 ,ul of Pharmacia stop mi.x. Samples were denatured at 90-95~C for S minutes (under this harsh condition, both DNA template and the dideoxy fragments are released 25 from the beads) and stored on ice prior to loading. A control experiment was performed in parallel using a 1 8-mer complementarv to the 3 ' end of target DNA as the sequencing primer instead of the duplex probe and the annealing of 18-mer to its target was carried out in a similar way as the annealing of the duplex probe.
Example 12 Chain Flon~tion of Tar~et Sequences.
Sequencing of immobilized target DNA can be performed with Sequenase Version 2Ø A total of S elongation reactions, one with each of 4 dideoxy nucleotides and one with all four simultaneously, are performed. A sequencing solution, cont~ining (40 mM Tris-HCI, pH 7.5, 20 mM MgCI2, and 50 mM NaCl, 10 mM dithiothreitol solution, 15 mM
sodium isocitrate and 10 mM MnCk, and 100 u/ml of Sequenase (1.5 units) is added to the hybridized target DNA. dATP, dCTP, dGTP and dTTP are added to 20 ~LM to initiate the elongation reaction. In the separate reactions, one of four ddNTP is added to reach a concentration of 8 ~lM. In the combined reaction all four ddNTP are added to the reaction to 8 ~M each.
The reaction mixtures were incubated at 0~C for S minutes room temperature for 5 minutes and 37~C for 5 minlltes. After the completion of extension, the supernatant was removed and the elongated DNA washed with 2 mM EDTA to terminate elongation reactions. Reaction products are analyzed by mass spectrometry.

Wo 96/32S04 PCTIUS9~105136 Fx~mI~le 13 Capillary Electrophoretic An~ysi~ of Tar~et Nucleic Acid.
Molecular weights oftarget sequences may also be determined by capillary electrophoresis. A single laser capillary eleckophoresis instrument can be used to monitor the performance of sample preparations 5 in high pclrolmance capillary electrophoresis sequencing. This instrument is designed so that it is easily converted to multiple channel (wavelengths) detection.
An individual element of the sample array may be engineered directly to serve as the sample input to a capillary. Typical capillaries are 10 250 rnicrons o.d. and 75 microns i.d. The sample is heated or denatured to release the DNA ladder into a liquid droplet. the silicon array surfaces is ideal for this purpose. The capillary can be brought into contact with the droplet to load the sample.
To facilitate loading of large numb~rs of samples 15 simultaneously or seqllenti~lly, there are two basic methods. With 250 micron o.d. capillaries it is feasible to match the dimensions of the target array and the capillary array. Then the two could be brought into contact manually or even by a robot arm using a jig to assure accurate alignment.
An electrode may be engineered directly into each sector of the silicon 20 surface so that sample loading would only require contact between the surface and the capillary array.
The second method is based on an inexpensive collection system to capture ~actions eluted from high performance capillary electrophoresis. Dilution is avoided by using designs which allow sample 25 collection without a perpendicular sheath flow. The same apparatus designed as a sample collector can also serve inversely as a sample loader.

In this case, each row of the sample array, equipped with electrodes, is used directly to load samples automatically on a row of capillaries. Using either method, sequence information is ~letermined and the target sequence constructed.
5 Example 14 Mass Spectrolnetry of NucleicAcids.
Nucleic acids to be analyzed by mass spectrometry were redissolved in ultrapure water (MilliQ, Millipore) using amounts to obtain a concentration of 10 pmoles/~ll as stock solution. An aliquot ( 1 ,ul) of this concentration or a dilution in ultrapure water was mixed with 1 ~1 of the 10 makix solution on a flat metal surface serving as the probe tip and dried with a fan using cold air. In some experiments, cation-ion exchange beads in the acid form were added to the mixture of matrix and sample solution to stabilize ions formed during analysis.
MALDI-TOF spectra were obtained on different commercial 15 instruments such as Vision 2000 (Finnigan-MAT), VG TofSpec (Fisons Instruments), LaserTec Research (Vestec). The conditions were linear negative ion mode with an acceleration voltage of 25 kV. Mass calibration was done externally and generally achieved by using defined peptides of appropriate mass range such as insulin, gramicidin S, trypsinogen, bovine 20 serum albumen and cytochrome C. All spectra were generated by employing a nitrogen laser with 5 nanosecond pulses at a wavelength of 33 7 nrn. Laser energy varied between 1 o6 and 107 W/cm2. To improve signal-to-noise ratio generally, the intensities of 10 to 30 laser shots were accumulated. The output of a typical mass spectrometry showing 25 discrimination between nucleic acids which differ by one base is shown in Figure 14.

WO 96/32504 PC~/US96/OS1:}6 Example 15 Sequel-ce Determin~tion from M~ Speckornet~
Elongation of a target nucleic acid, in the presence of dideoxy chain termin~ting nucleotides, generated four families of chain-te~nin~ted fr~ nt~. The mass difference per nucleotide addition is 289.19 for dpC, 5 313.21 for dpA, 329.21 for dpG and 304.20 for dpT, respectively.
Comparison of the mass differences measured between fragments with the known masses of each nucleotide the nucleic acid sequence can be determined. Nucleic acid may also be sequenced by performing polymerase chain elongation in four separate reactions each with one dideoxy chain 10 tPrmin~tingnucleotide. To examine mass differences, 13 oligonucleotides from 7 to 50 bases in length were analyzed by MALDI-TOF mass spectrometry. The correlation of calculated molecular weights of the ddT
fragments of a Sanger sequencing reaction and their experimentally verified weights are shown in Table 10. When the mass spectrometry data from all 15 four chain termination reactions are combined, the molecular weight difference between two adjacent peaks can be use to determine the sequence.

WO 96t32504 PCT~US96105136 Table 10 Sl~mm~ry of Molecular Weights Expected v. Measured Fr~nent ~-mer) Calculated Mass Fx~?erirnental Mass Difference 7-mer 2104.45 2119.9 +15.4 10-mer 3011.04 3026.1 +15.1 11-mer 3315.24 3330.1 +14.9 19-mer 5771.82 5788.0 +16.2 20-mer 6076.02 6093.8 +17.8 24-mer 7311.82 7374.9 +63.1 26-mer 7945.22 7960.9 +15.7 33-mer 10112.63 10125.3 +12.7 37-mer 11348.43 11361.4 +13.0 38-mer 11652.62 11670.2 +17.6 42-mer 12872.42 12888.3 +15.9 46-mer 14108.22 14125.0 +16.8 50-mer 15344.02 15362.6 +18.6 Example 16 Reduced Pass Sequencin~-To maximize the use of PSBH arrays to produce Sanger ladders, the sequence of a target should be covered as completely as possible with the lowest amount of initial sequencing redundancy. This will maximize the performance of individual elements of the arrays and m~Ximi7e the amount of useful sequence data obtained each time an array is used. With an unknown DNA, a full array of 1024 elements (Mwo I or BsiYI cleavage) or 256 elements (TspR I cleavage) is used. A 50 kb target DNA is cut into about 64 fragments by Mwo I or BsiYI or 30 fragments by ~spR I, respectively. Each fragment has two ends both of which can be captured independently. The coverage of each array after capture and ignoring degeneracies is 128/1024 sites in the first case and 60/256 sites in - the second case. Direct use of such an array to blindly deliver samples element by element for mass spectrometry sequencing would be inefficient since most array elements will have no sarnples.
In one method, phosph~t~ecl double-stranded targets are used at high concentrations to saturate each array element that detects a sample.
5 The target is ligated to make the caplule irreversible. Next a different sample mixture is exposed to the array and subsequently ligated in place.
This process is repeated four or five times until most of the elements of the array contain a unique sample. Any tandem target-target complexes will be removed by a subsequent ligating step because all of the targets are 1 0 phosphatased.
Alternatively, the array may be monitored by confocal microscopy after the elongation reactions. This reveals which elements contain elongated nucleic acids and this information is communicated to an automated robotic system that is l-ltim~tely used to load the samples onto a 15 mass speckometry analyzer.
Example 17 Synthesis of Mass Modified Nucleic Acid Primers.
Mass modification at the 5' sugar: Oligonucleotides were synthesized by standard automated DNA synthesis using ~-cyanoethylphosphoamidites and a 5'-amino group introduced at the end of 20 solid phase DNA synthesis. The total amount of an oligonucleotide synthesis, starting with 0.25 micromoles CPG-bound nucleoside, is deprotected with concentrated aqueous ammonia, purified via OligoPAKTM
Cartridges (Millipore; Bedford, MA) and Iyophilized. This material with a 5'-terminal amino group is dissolved in 100 ~11 absolute N, N-25 dimethylformamide (DMF) and condensed with 10 ,umole N-Fmoc-glycine pentafluorophenyl ester for 60 minutes at 25 ~C. After ethanol precipitation WO 96132504 PCTJUS96,'0S136 and centrifugation, the Fmoc group is cleaved off by a 10 minute treatment with 100 ~Ll of a solution of 20% piperidine in N,N-dimethylforrnamide.
, Excess piperidine, DMF and the cleavage product from the Fmoc group are removed by ethanol precipitation and the precipitate lyophilized from 10 5 mM TEAA buffer pH 7.2. This material is now either used as primer for ~e Sanger DNA sequencing reactions or one or more glycine residues (or other suitable protected amino acid active esters) are added to create a series of mass-modified primer oligonucleotides suitable for Sanger DNA or RNA
sequencing.
10Mass modification at the heterocyclic base with glycine:
Starting material was 5-(3-aminopropynyl-1)-3'5'-di-p-tolyldeoxyuridine prepared and 3' 5'-de-O-acylated (Haralambidis et al., Nuc. Acids Res.
15 :4857-76, 1987). 0.281 g (1.0 mmol) 5-(3-aminopropynyl-1)-2'-deoxyuridine were reacted with 0.927 g (2.0 mmol) N-Fmoc-glycine 15 pentafluorophenylester in 5 ml absolute N,N-dimethylformamide in the presence of 0.129g (1 mmol; 174 1ll) N,N-diisopropylethylamine for 60 minll1es at room temperature. Solvents were removed by rotary evaporation and the product was purified by silica gel chromatography (Kieselgel 60, Merck; column: 2.5 x 50 cm, elution with chloroform/methanol mixtures).
20 Yield was 0.44 g (0.78 mmol; 78%). To add another glycine residue, the Fmoc group is removed with a 20 minutes treatment with 20% solution of piperidine in DMF, evaporated in vacuo and the remaining solid material extracted three times with 20 ml ethylacetate. After having removed the remaining ethyl~cet~te, N-Fmoc-glycine pentafluorophenylester is coupled 25 as described above.5-(3(N-Fmoc-glycyl)-amidopropynyl-1)-2'-deoxyuridine is transformed into the 5'-O-dimethoxytritylated nucleoside-3'-O-~-WO 96/32504 PCT/US~6/OS136 cyanoethyl-N,N-diisopropylphosphoamidite and incorporated into automated oligonucleotide syn~esis. This glycine modified thymidine analogue building block for chemical DNA synthesis can be used to substitute one or more of the thymidine/uridine nucleotides in the nucleic S acid primer sequence. The Fmoc group is removed at the end of the solid phase synthesis with a 20 minute treatment with a 20% solution of piperidine in DMF at room temperature. DMF is removed by a washing step with acetonitrile and the oligonucleotide deprotected and purified.
Mass modification at the heterocyclic base with ~ ol~nine 10 0.281 g (1.0 -mmol) 5-(3-Aminopropynyl-1)-2'-deoxyuridine was reacted with N-Fmoc-~-alanine pentafluorophenylester (0.955 g; 2.0 rnmol) in 5 ml N,N-dimethylformamide (DMF) in the presence of 0.129 g (174 ~11; 1.0 mmol) N,N-disopropylethylamine for 60 minutes at room temperature.
Solvents were removed and the product purified by silica gel 15 chromatography. Yield was 0.425 g (0.74 mmol; 74%). Another ~-alanine moiety can be added in exactly the same way after removal of the Fmoc group. The ~el~a~ion of the S'-O-dimethoxytritylated nucleoside-3'-O-~-cyanoethyl-N,N-diisopropylphosphoamidite from 5-(3-(N-Fmoc-~-alanyl)-amidopropynyl-1)-2'-deoxyuridine and incorporation into automated 20 oligonucleotide synthesis is performed under standard conditions. This building block can substitute for any of the thymidine/uridine residues in the nucleic acid primer sequence.
Mass modification at the heterocyclic base with ethylene monomethyl ether: 5-(3-aminopropynyl-1)-2'-deoxyuridine was used as a 25 nucleosidic component in this example. 7.61 g (100.0 mmol) freshly distilled ethylene glycol monomethyl ether dissolved in 50 ml absolute wo 96132~04 PcTruss6Josl36 pyridine was reacted with 10.01 g (100.0 mmol) recryst~lli7e~1 succinic anhydride in the presence of 1.22 g (10.0 mmol) 4-N,N--dimethylaminopyridine overnight at room temperature. The reaction was terrnin~1 by the addition of water (5.0 ml), the reaction mixture evaporated 5 in vacuo, co-evaporated twice with dry toluene (20 ml each) and the residue redissolved in 100 ml dichloromethane. The solution was twice extracted sllccessively with 10% aqueous citric acid (2 x 20 ml) and once with water (20 ml) and the organic phase dried over anhydrous sodium sulfate. The organic phase was evaporated in vacuo. Residue was redissolved in 50 ml 10 dichloromethane and preci~i~led into 500 ml pentane and the precipitate dried in vacuo. Yield was 13.12 g (74.0 mmol; 74%). 8.86 g (50.0 mmol) of succinylated ethylene glycol monomethyl ether was dissolved in 100 ml dioxane cont~ining 5% dry pyridine (5 ml) and 6.96 g (50.0 mmol) 4-nitrophenol and 10.32 g (50.0 mmol) dicyclohexylcarbodiimide was added 15 and the reaction run at room temperature for 4 hours. Dicyclohexylurea was removed by filtration, the filtrate evaporated in vacuo and the residue redissolved in 50 ml anhydrous DMF. 12.5 ml (about 12.5 mmol 4-nitrophenylester) ofthis solution was used to dissolve 2.81 g (10.0 mmol) 5-(3-aminopropynyl-1)-2'-deoxyuridine. The reaction was performed in the 20 presence of 1.01 g (10.0 mmol; 1.4 ml) triethylamine overnight at room tempcl~Lure. The reaction mixture was evaporated in vacuo, co-evaporated with toluene, redissolved in dichloromethane and chromatographed on silicagel (Si60, Merck; column 4 x 50 cm) with dichloromethane/methanol mixtures. Fractions containing the desired compound were collected, 25 evaporated, redissolved in 25 ml dichloromethane and precipitated into 250 ml pentane. The dried precipitate of 5-(3-N-(O-succinyl ethylene glycol monomethyl ether)-amidopropynyl-1)-2'-deoxyuridine (yield 65%) is 5'-O-dimethoxytritylated and transformed into the nucleoside-3'-O-~-cyanoethyl-N, N-diisopropylphosphoamidite and incorporated as a building block in the automated oligonucleotide synthesis according to standard procedures. The 5 mass-modified nucleotide can substitute for one or more of the thymidine/uridine residues in the nucleic acid primer sequence.
Deprotection and purification of the primer oligonucleotide also follows standard procedures.
Mass modification at the heterocyclic base with diethylene 10 glycol monomethyl ether: Nucleosidic starting material was as in previous examples, 5-(3-aminopropynyl-1)-2'-deoxyuridine. 12.02 g (100.0 mmol) freshly distilled diethylene glycol monomethyl ether dissolved in 50 ml absolute pyridine was reacted with 10.01 g (100.0 mmol) recrystallized succinic anhydride in the presence of I .22 g ( 10.0 mmol) 4-N, N-15 dimethylaminopyridine (DMAP) overnight at room temperature. Yield was18.35 g (82.3 mmol; 82.3%). 11.06 g (50.0 mmol) of succinylated diethylene glycol monomethyl ether was transformed into the 4-nitrophenylester and, subsequently, 12.5 mmol was reacted with 2.81 g ( 10.0 mmol) of 5-(3-aminopropynyl -1)-2'-deoxyuridine. Yield after silica gel 20 column chromatography and precipitation into pentane was 3.34 g (6.9 mmol; 69%). After dimethoxytritylation and transformation into the nucleoside-~-cyanoethylphosphoamidite, the mass-modified building block is incorporated into automated chemical DNA synthesis. Within the sequence of the nucleic acid primer, one or more of the thymidine/uridine 25 residues can be substituted by this mass-modified nucleotide.

wo 96/32504 PcTruss6l0sl36 Mass Modification at the heterocyclic base with glycine:
Starting material was N6-benzoyl-8-bromo-5'-0-(4,4'-dimethoxytrityl)-2'-deox~yadenosine (Singh et al., Nuc. Acids Res.18:3339-45,1990). 632.5 mg (1.0 mmol) ofthis 8-bromo-deoxyadenosine derivative was suspended in 5 5 ml absolute ethanol and reacted with 251.2 mg (2.0 mmol) glycine methyl ester (hydrochloride) in ~e presence of 241.4 mg (2.1 mmol; 366 ,ul) N,N-diisopropylethylamine and refluxed until the starting nucleosidic material had disappeared (4-6 hours) as checked by thin layer chromatogranhy (TLC). The solvent was evaporated and the residue purified by silica gel 10 chromatography (column 2.5 x 50 cm) using solvent mixtures of chloroform/methanol cont~inin~ 0.1% pyridine. Product fractions were combined, the solvent evaporated, the fractions dissolved in 5 ml dichloromethane and precipitated into 100 ml pentane. Yield was 487 mg (0.76 mmol; 76%). Transformation into the corresponding nucleoside-~-15 cyanoethylphospho amidite and integration into automated chemical DNAsynthesis is performed under standard conditions. During final deprotection with aqueous concentrated ammonia, the methyl group is removed from the glycine moiety. The mass-modified building block can substitute one or more deoxyadenosine/adenosine residues in the nucleic acid primer 20 sequence.
Mass modification at the heterocyclic base with glycylglycine: 632.5 mg (1.0 mmol) N6-Benzoyl-8-bromo-5'-O-(4,4'dimeethoxytrityl)2'-deoxyadenosine was suspended in 5 ml absolute ethanol and reacted with 324.3 mg (2.0 mmol) glycyl-glycine methyl ester 25 in the presence of 241.4 mg (2.1 mmol; 366,~1) N, N-diisopropylethylamine.
The mixture was refluxed and completeness of the reaction checked by WO 96132504 PCI~/US96/OS136 TLC. Yield after silica gel column chromatography and precipitation into pentane was 464 mg (0.65 mmol; 65%). Transformation into the nucleoside-~-cyanoethylphosphoamidite and into synthetic oligonucleotides is done according to standard procedures.
S Mass Modi~lcation at the heterocyclic base with glycol monomethyl ether: Starting material was 5'-0-(4,4-dimethoxytrityl)-2'-amino-2'-deoxythymidine synthesized (Verheyden et al., J. Org. Chem.
36:250-54, 1971; Sasaki et al, J. Org. Chem. 41:3138-43, 1976; Imazawa et al., J. Org. Chem. 44:2039-41, 1979; Hobbs et al., J. Org. Chem. 42:714-19, 1976; Ikehara et al., Chem. Pharm. Bull. Japan 26:240-44, 1978). 5'-0-(4,4-Dimethoxytrityl)-2'-amino-2'-deoxythymidine (559.62 mg; 1.0 mmol) was reacted with 2.0 mmol of the 4-nitrophenyl ester of succinylated ethylene glycol monomethyl ether in 10 ml dry DMF in the presence of 1.0 mmol (140 ,ul) triethylamine for 18 hours at room tempeldlule. The reaction mixture was evaporated in vacuo, co-evaporated with toluene, redissolved in dichloromethane and purified by silica gel chromatography (Si60, Merck;
column: 2.5 x 50 cm; eluent: chloroform/methanol mixtures containing 0.1 %
triethylamine). The product containing fractions were combined, evaporated and precipitated into pentane. Yield was 524 mg (0.73 mmol; 73%).
Transformation into the nucleoside-~-cyanoethyl-N,N--diisopropylphosphoamidite and incorporation into the automated chemical DNA synthesis protocol is performed by standard procedures. The mass-modified deoxythymidine derivative can substitute for one or more of the thymidine residues in the nucleic acid primer.
In an analogous way, by employing the 4-nitrophenyl ester of succinylated diethylene glycol monomethyl ether and triethylene glycol monomethyl ether, the corresponding mass-modified oligonucleotides are l)r~aled. In the case of only one incorporated mass-modified nucleoside within the sequence, the mass difference between the ethylene, diethylene and triethylene glycol derivatives is 44.05, 88.1 and 132.15 daltons, 5 respectively.
Mass modification at the heterocyclic base by allylation:
Phosphorothioate-c(mtAining oligonucleotides were prepared (Gait et al., Nuc. Acids Res. 19:1183, 1991). One, several or all internucleotide linkages can be modified in this way. The (-)M13 nucleic acid primer sequence (17-10 mer) 5'-dGTAAAACGACGGCCAGT (SEQ ID NO 29) is synthesized in 0.25 ,umole scale on a DNA synthesizer and one phosphorothioate group introduced after the final synthesis cycle (G to T coupling). Sulfurization, deprotection and purification followed standard protocols. Yield was 3 l .4 nmole ( 12.6% overall yield), corresponding to 31.4 nmole phosphorothioate 15 groups. Alkylation was performed by dissolving the residue in 31.4 ,ul TE
buffer (0.01 M Tris pH 8.0, 0.001 M EDTA) and by adding 16 ,ul of a solution of 20 mM solution of 2-iodoethanol (320 nmole; 10-fold excess with respect to phosphorothioate diesters) in N,N-dimethylformamide (DMF). The alkylated oligonucleotide was purified by standard reversed 20 phase HPLC (~P-18 Ultraphere, BeckmAn; column: 4.5 x 250 mm; 100 mM
triethyl ammonium A~etAte, pH 7.0 and a gradient of 5 to 40% acetonitrile).
In a variation of this procedure, the nucleic acid primer contAining one or more phosphorothioate phosphodiester bond is used in the Sanger sequencing reactions. The primer-extension products of the four 25 sequencing reactions are purified, cleaved offthe solid support, Iyophilized and dissolved in 4 ,ul each of TE buffer pH 8.0 and alkylated by addition of CA 022l8l88 l997- lO- l4 WO 96t32504 PCTtUS96/05136 2 ,~1 of a 20 mM solution of 2-iodoethanol in DMF. It is then analyzed by ES and/or MALDI mass spectrometry.
In an analogous way, employing instead of 2-iodoethanol, e.g., 3iodopropanol, 4-iodobutanol mass-modified nucleic acid primer are 5 obtained with a mass difference of 14.03, 28.06 and 42.03 daltons respectively compared to the unmodified phosphorothioate phosphodiester-containing oligonucleotide.
Example 18 Mass Modification of Nucleotide Triphosphates.

WO 96/32S04 PCT/~JS96/OS136 Mass m~slifi~ of nucleoffde triphosphates at the 2' and 3'aminofunction: Startingmaterialwas2'-azido-2'-deoxyuridine~ d according to literature (Verheyden et al., J. Org. Chem. 36:250, 1971), which was 4,4- dimethoxytritylated at 5'-OH with 4,4-dimethoxytrityl S chloride in pyridine and acetylated at 3'-OH with acetic anhydride in a one-pot reaction using standard reaction conditions. With 191 mg (0.71 mmol) 2'-azido-2'-deoxyuridine as starting material, 396 mg (0.65 mmol; 90.~%) 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-azido-2'-deoxyuridine was obtained after purification via silica gel chromatography. Reduction of the 10 azido group was performed (Barta et al., Tetrahedron 46:587-94, 1990).
Yield of 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'-deoxyuridine after silica gel chromatography was 288 mg (0.49 mmol; 76%). This protected 2'-amino-2'-deoxyuridine derivative (588 mg, 1.0 mmol) was reacted with 2 equivalents (927 mg; 2.0 mmol) N-Fmoc-glycine 15 pentafluorophenyl ester in 10 ml dry DMF overnight at room temperature in the presence of 1.0 mmol (174,ul) N,N-diisopropylethylamine. Solvents were removed by evaporation in vacuo and the residue purified by silica gel chromatography. Yield was 711 mg (0.71 mmol; 82%). Detritylation was achieved by a one hour treatment with 80% aqueous acetic acid at room 20 temperature. The residue was evaporated to dryness, co-evaporated twice with toluene, suspended in 1 ml dry acetonitrile and 5'-phosphorylated with POCI3 and directly transformed in a one-pot reaction to the 5'-triphosphate using 3 ml of a 0.5 M solution (1.5 mmol) tetra (tri-n-butylammonium) pyrophosphate in DMF according to literature. The Fmoc and the 3'-O-25 acetyl groups were removed by a one-hour treatment with concentrated aqueous ammonia at room temperature and the reaction mixture evaporated WO ~6132S04 PCT/US96105136 g8 and lyophili7e~1 Purification also followed standard procedures by using anion-exch~n~e chromatography on DEAE Sephadex with a linear gradient of ~iethylammoniurn bicarbonate (0.1 M - 1.0 M). Triphosph~te colltS~ g fractions, checked by thin layer chromatography on polyethyleneimine S cellulose plates, were collected, evaporated and Iyophili7ed Yield by W-absorbance of the uracil moiety was 68% or 0.48 mmol.
A glycyl-glycine modified 2'-amino-2'-deoxyuridine-5'-triphosphate was obtained by removing the Fmoc group from 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-N(N-9-fluorenylmethyloxycarbonyl-glycyl)-2'-amino-2'-deoxyuridine by a one-hour treatment with a 20% solution of piperidine in DMF at room t~ e.dlure, evaporation of solvents, two-fold co-evaporation with toluene and subsequent condensation with N-Fmoc-glycine pentafluorophenyl ester. Starting with 1.0 mmol of the 2'-N-glycyl-2'-amino-2'-deoxyuridine derivative and following the procedure described above, 0.72 mmol (72%) of the corresponding 2'-(N-glycyl-glycyl)-2'-amino-2'-deoxyuridine-5'triphosphate was obtained.
Startingwith 5'-0-(4,4-dimethoxytrityl)-3'-O-acetyl-2'-amino-2'deoxyuridine and coupling with N-Fmoc-~-alanine pentafluorophenyl ester, the corresponding 2'-(N-~-alanyl)-2'-amino-2'-deoxyuridine-5'-triphosphate are synthesized. These modified nucleoside triphosphates are incorporated during the Sanger DNA sequencing process in the primer-extension products. The mass difference between the glycine, ~-alanine and glycyl-glycine mass-modified nucleosides is, per nucleotide incorporated, 58.06, 72.09 and 115.1 daltons, respectively.
When startingwith 5'-0-(4,4-dimethoxytrityl)-3'-amino-2',3' 1 -dideoxythymidine, the corresponding 3'-(N-glycyl)-3'-amino-,3'-(-N-glycyl-WO 96/32504 PCIJUS96S~S136 glycyl)-3'-amino-, and 3'~ -ala~yl)-3'-amino-2',3'-dideoxythymidine-5'-triphosphates can be obtained. These mass-modified nucleoside triphosphates serve as a terrnin~ting nucleotide unit in the Sanger DNA
sequencing reactions providing a mass difference per tçrrnin~ted fragment 5 of 58.06, 72.09 and 115.1 daltons respectively when used in the multiplexing sequencing mode. The mass-differentiated fragments are analyzed by ES and/or MALDI mass spectrometry.
Mass mo~lifi~ n of nucleotide triphosphates at C-5 of the heterocyclic base: 0.281 g (1.0 mmol) 5-(3-Aminopropynyl-1)-2'-10 deoxyuridine was reacted with either 0.927 g (2.0 mmol) N-Fmoc-glycine pentafluorophenylester or 0.955g (2.0 mmol) N-Fmoc-~-alanine pentafluorophenyl ester in 5 ml dry DMF in the presence of 0.129 g N, N-diisopropylethylamine (174 ~11, 1.0 mmol) overnight at room temperature.
Solvents were removed by evaporation in vacuo and the condensation 15 products purified by flash chromatography on silica gel (Still et al., J. Org., Chem. 43: 2923-25, 1978). Yields were 476 mg (0.85 mrnol; 850%) for the glycine and 436 mg (0.76 mmol; 76%) for the ~-alanine derivatives. For the synthesis of the glycyl-glycine derivative, the Fmoc group of 1.0 mmol Fmoc-glycine-deoxyuridine derivative was removed by one-hour treatment 20 with 20% piperidine in DMF at room temperature. Solvents were removed by evaporation in vacuo, the residue was coevaporated twice with toluene and condensed with 0.927 g (2.0 mmol) N-Fmoc-glycine pentafluorophenyl ester and purified as described above. Yield was 445 mg (0.72 mmol; 72%).
The glycyl-, glycyl-glycyl- and ~-alanyl-2'-deoxyuridine derivatives, N-25 protected with the Fmoc group were transformed to the 3'-O-acetyl derivatives by tritylation with 4,4-dimethoxytrityl chloride in pyridine and acetylation with acetic anhydride in pyridine in a one-pot reaction and subsequently detritylated by one hour treatment with 80% aqueous acetic acid according to standard procedures. Solvents were removed, the residues dissolved in 100 ml chloroform and extracted twice with 50 ml 10% sodium 5 bicarbonate and once with 50 ml water, dried with sodium sulfate, the solvent evaporated and the residues purified by flash chromatography on silica gel. Yields were 361 mg (0.60 mmol; 71%) for the glycyl-, 351 mg (0.57 rnrnol; 75%) for the ~-alanyl- and 323 mg (0.49 mmol; 68%) for the glycyl-glycyl-3-0'-acetyl-2'-deoxyuridine derivatives, respectively.
10 Phosphorylation at the 5'-OH with POCI3, transformation into the 5'-triphosphate by in situ reaction with tetra(tri-n-butylammonium) pyrophosphate in DMF, 3'-de-0-acetylation, cleavage of the Fmoc group, and final purification by anion-exchange chromatography on DEAE-Sephadex was perforrned and yields according to W-absorbance of the 15 uracil moiety were 0.41 mmol 5-(3-(N-glycyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (84%), 0.43 mmol 5-(3-(N-~-alanyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (75%) and 0.38 mmol 5-(3-(N-glycyl-glycyl)-amidopropynyl-1)-2'-deoxyuridine-5'-triphosphate (78%). These mass-modified nucleoside triphosphates were incorporated 20 during the Sanger DNA sequencing primer-extension reactions.
When using 5-(3-aminopropynyl)-2',3'-dideoxyuridine as starting material and following an analogous reaction sequence the corresponding glycyl-, glycyl-glycyl-and ~-alanyl-2',3'-dideoxyuridine-5'-triphosphates were obtained in yields of 69%, 63% and 71 %, respectively.
25 These mass-modifled nucleoside triphosphates serve as chain-termin~ting nucleotides during the Sanger DNA sequencing reactions. The mass-WO 96J325~4 PCTIUS96/05136 modified sequencing ladders are analyzed by either ES or MALDI mass spectrometry.
, Mass modification of nucleotide triphosphates: 727 mg (1.0 mmol) of N6-(4-tert-butylphenoxyacetyl)-8-glycyl-5'-(4,4--5 dime~oxytrityl)-2'- deoxyadenosine or 800 mg (1.0 mmol) N6-(4-tert-butylphenoxyacetyl)-8-glycyl-glycyl-5'-(4,4-dimethoxytrityl)-2'-deoxyadenosine ~l~cd according to literature (Koster et al., Tetrahedron 37:362, 1981) were acetylated with acetic anhydride in pyridine at the 3'-OH, detritylated at the 5'-position with 80% acetic acid in a one-pot reaction 10 and transformed into the 5'-triphosphates via phosphorylation with POCI3 and reaction in situ with tetra(tri-n-butylamrnonium) pyrophosphate.
Deprotection ofthe N6 tert-butylphenoxyacetyl, the 3'-O-acetyl and the O-methyl group at the glycine residues was achieved with concentrated aqueous ammonia for ninety minutes at room temperature. Ammonia was 15 removed by Iyophilization and the residue washed with dichloromethane, solvent removed by evaporation in vacuo and the remaining solid material purified by anion-exchange chromatography on DEAE-Sephadex using a linear gradient of triethylammonium bicarbonate from 0.1 to 1.0 M. The nucleoside triphosphate cont~ining fractions (checked by l~C on 20 polyethyleneimine cellulose plates) were combined and Iyophili7e~1 Yield of the ~-glycyl-2'-deoxyadenosine-5'-triphosphate (determined by W-absorbance of the adenine moiety) was 57% (0.57 mmol). The yield for the 8-glycyl-glycyl-2'-deoxyadenosine-5'-triphosphate was 51% (0.51 mmol).
These mass-modified nucleoside triphosphates were incorporated during 25 primer-extension in the Sanger DNA sequencing reactions.

When using the corresponding N6-(4-tert-butylphenoxyacetyl)-8-glycyl- or-glycyl-glycyl-5'-0-(4,4-dimethoxytrityl)-2',3'-dideoxyadenosine derivatives as startirlg materials (for the introduction ofthe 2',3'-fimction: Seela et al., Helvetica Chimica Acta 74: 1048-58, 1991).
S Using an analogous reaction sequence, the chain-tçnnin~tin~ mass-modified nucleoside triphosphates 8-glycyl- and 8-glycyl-glycyl-2'.3'-dideoxyadenosine-5'-triphosphates were obtained in 53 and 47% yields, respectively. The mass-modified sequencing fragment ladders are analyzed by either ES or MALDI mass spectrometry.
10 Example 19 ~ Modification of Nucleotides by Alkylation A~er Sanger Sequencin~
2',3'-Dideoxythymidine-5'-(alpha-S)-triphosphate was prepared according to published procedures (for the alpha-S-triphosphate moiety: Eckstein et al., Biochemistry 15: 1685, 1976) and Accounts Chem.
15 Res. 12:204, 1978) and for the 2',3'-dideoxy moiety: Seela et al., Helvetica Chimica Acta 7~:1048-58, 1991). Sanger DNA sequencing reactions employing 2'-deoxythymidine-5'-(alpha-S)-triphosphate are performed according to standard protocols. When using 2',3'-dideoxythymidine-5'-(alpha-S)-triphosphates, this is used instead of the unmodified 2',3'-20 dideoxythymidine-5'-triphosphate in standard Sanger DNA sequencing. The template (2 picomole) and the nucleic acid M13 sequencing primer (4 picomole) are annealed by heating to 65~C in 100 1ll of 10 mM Tris-HCl~
pH 7.5, 10 mM MgCl2, 50 mM NaCl, 7 mM dithiothreitol (DTT for 5 minutes and slowly brought to 37~C during a one hour period. The 25 sequencing reaction mixtures contain, as exemplified for the T-specific termination reaction, in a final volume of 150 1ll, 200 ~lM (final WO 96/3251)4 PcTJus96Jo5136 conc~ lion) each of dATP, dCTP, ~ , 300 ~lM c7-deaza-dGTP, S IlM
2',3'dideoxythymidine-5'-(alpha-S)-triphosphate and 40 units Sequenase.
Polymerization is pe~ro~ ed for 10 minutes at 37~C, the reaction mixture heated to 70 ~C to inactivate the Sequenase, ethanol precipitated and coupled 5 to thiolated Sequelon membrane disks (8 mm diameter). Alkylation is performed by treating the disks with 10 ~Ll of 10 mM solution of either 2-iodoethanol or 3-iodopropanol in NMM (N-methylmorpholine/water/2-propanol, 2/49/49, v/v/v) (three times), washing with 10 ~11 NMM (three times) and cleaving the alkylated T-termin~ted primer-extension products 10 off the support by tre~ttnent with DTT. Analysis of the mass-modified fragment families is performed with either ES or MALDI mass spectrometry.
Example 20 Mass Modification of an Oligonucleotide.
This method, in addition to mass modification, also modifies 15 the phosphate backbone of the nucleic acids to a non-ionic polar form.
Oligonucleotides can be obtained by chemical synthesis or by enzymatic synthesis using DNA polymerases and a-thio nucleoside triphosphates.
This reaction was performed using DMT-TpT as a starting material but the use of an oligonucleotide with an alpha thio group is also 20 al~p~ liate. For thiolation, 45 mg (0.05 mM) of compound 1 (Figure 15), is dissolved in 0.5 ml acetonitrile and thiolated in a 1.5 ml tube with 1.1-diozo-l-H-benzo[1,2]dithio-3-on (Beaucage reagent). The reaction was allow to proceed for 10 minlltes and the produce is concentrated by thin layer chromatography with the solvent system dichloromethane/96%
25 ethanol/pyridine (87%/13%/1%; v/v/v). The thiolated compound 2 (Figure 15) is deprotected by treatment with a mixture of concentrated aqueous ammonia/acetonitrile (1/1; v/v) at room temperature. This reaction is monitored by thin layer chromatography and the qu~ e removal of the beta-cyanoethyl group was accomplished in one hour. This reaction mixture was evaporated in vacuo.
To synthesize the S-(2-amino-2-oxyethyl)thiophosphate triester of DMT-TpT (compound 4), the foam obtained after evaporation of the reaction mixture (compound 3) was dissolved in 0.3 ml acetonitrile/pyridine (5/1; v/v) and a 1.5 molar excess of iodoacetamide added. The reaction was complete in 10 minutes and the precipitated salts 10 were removed by centrifugation. The supernatant is lyophilized, dissolved in 0.3 ml acetonitrile and purified by preparative thin layer chromatography with a solution of dichloromethane/96% ethanol (85%/15%; v/v). Two fractions are obtained which contain one of the two diastereoisomers. The two forms were separated by HPLC.
15 Example 21 MALDI-MS Analysis of a Mass-Modified Oligonucleotide.
~ 1 7-mer was mass modified at C-5 of one or two deoxyuridine moieties. 5-[1 3-(2-Methoxyethoxyl)-tridecyne- I -yl]-5 '-O-(4,4 ' -dimethoxytrityl)-2 ' -deoxyuridine-3 '-~-cyanoethyl-N,N-diisopropylphosphoamidite was used to synthesize the modified 1 7-mers.

Wo 96/32504 pcTruss6Josl36 The modified 17-mers were:

5d~AAAACGACGGCCAGUG) (molecul~m~s:5454) (SEQIDNO30) X X
d(UAAAACGCGGCCAGUG) (molecul~ m~s 5634) (SEQ IDNO 31) where X = -C=C-(CH2)"-OH
(nnmorlifi~d 17-mer: molecularmass: 5273) The samples were ~ ed and 500 fmol of each modified 17-15 mer was analyzed using MALDI-MS. Conditions used were reflectron positive ion mode with an acceleration of S kV and post-acceleration of 20 kV. The MALDI-TOF spectra which were generated were superimposed and are shown in Figure 16. Thus, mass modification provides a distinction detectable by mass spectrometry which can be used to identify base 20 sequence information.

Example 22 Capture and Sequencing of a Double-Stranded Target Nucleic Acid.
In another experiment, a nucleic acid was captured and 25 sequenced by strand-displacement polymerization. This reaction is shown schem~tically in Figure 17. Double-stranded DNA target was prepared by PCR and attached to magnetic beads as described in Example 6. EcoR I
digested plasmid NB34 was used as the DNA template for amplification.
NB34 comprises a PCRTM II plasmid (Invitrogen) with a one kb target 30 human DNA insert. PCR was performed with an 16-nucleotide upstream ~ primer (primer I, 5'-AACAGCTATFACCATG-3'; SEQ ID NO. 32), and a downstream 5 -end biotinylated 1 8-nucleotide primer (primer II, 5'-biotin-CTGAATTAGTCAGGTTGG-3', SEQ ID NO. 33). Five hundred basepair PCR products, cont~inin~ a single BstX I site, were immobilized by çhment to magnetic beads which were resuspended in a total of 300 ~11 ?
reaction buffer cont~ining 200 units of BstX I restriction endonuclease S (Boehringer Mannheim; Tntli~n~polis, IN), 50 mM Tris-HCl pH 7.5, 10 mM
MgCl2, 100 mM NaCl and 1 mM dithiothreitol. The mixture was incubated at 45~C for three hours or until digestion was complete which was monitored by agarose gel electrophoresis. After digestion, magnetic beads were washed twice with 300 ~11 of TE to remove digested and non-immobilized fragments, excess nucleotides and restriction endonuclease.
This immobilized DNA was dephosphorylated by resuspending the beads in 100 ,ul buffer (500 mM Tris-HCl, pH 9.0, 1 mM
MgCl2, 0.1 mM ZnCl2, and 1 mM spermidine) containing five units of calf intes~in~l alkaline phosphatase (Promega; Madison, WI). The reaction was incubation at 37~C for 15 minutes and at 56~C for 15 minutes. Five additional units of calf intestinal alkaline phosphatase was added and a second incubation was performed at 37~C for 15 minutes and at 56~C for 15 minl~tes. Beads were washed twice with TE and resuspended in 300 ~11 of fresh TE containing 1 M NaCI.
Loading of the beads was checked by incubating 10 1ll of the beads with 10 ~1 of formamide at 95~C for 5 minutes (or by boiling in TE).
The mixture was analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. A 10 ~1 bead aliquot generally contains about 80 ng of immobilized double stranded DNA.
A partial duplex DNA probe containing a four base 3 ' overhang was used as a sequencing primer and was ligated with BstX I

WO 96t32504 1~CTJUS96J~5136 digested DNA fr~ ont~ which were immobilized on magnetic beads. The partial duplex had a 5'-fluorescein labeled 23 mer (DF25-SF) cont~ininp: a 5' base paring region and a 4-base 3' single stranded region (which is complementary to the sequence of the 5'-protruding end of the S corresponding BstXI digested target DNA as l)r~al ed above and a 19 mer (G-CMl) complementary to the base pairing region ofthe 23 mer. The 19 mer was 5' phosphorylated by the T4 DNA Polymerase and annealed to the corresponding 23 mer in TE at the same molar ratio. Beads, prepared from ~lk~line phosphatase treatment which have about 10 pmol immobilized 10 DNA template, were ligated to 25 pmol of partially duplex probe in an 100 ~11 volume cont~ining 200 units of T4 DNA ligase (New Fngl~n(l Biolabs;
Beverly, MA), 50 mM Tris-HCI, pH 7.8, 10 mM MgCI2, 10 mM
dithiothreitol, 1 mM ATP, 25 ,ug/ml bovine serum albumin. Ligation reactions were pelrollned at room temperature for two hours or 4~C
15 overnight. Beads were washed twice with TE and resuspended in 300 ~LI of the same buffer.
Sequencing reactions: Thirty ,ul of beads containing the ligation product were used for each sequencing reaction. Beads were resuspended in a 13 ~11 volume containing 1.5 ~11 of 10 x Klenow buffer (100 20 mM Tris-HC1, pH 7.5, 50 mM MgCI2, and 75 mM dithiothreitol) and with or without one ~1 of single stranded DNA binding protein (SSB, S llg/,ul;
USB; Cleveland, Ohio). Mixtures were incubated on ice for 5 minutes followed with the addition of 5 units of Klenow Fragment (New England Biolabs). The reaction volume was split into four termination mixes, each 25 consisting of I ~I DMSO and 3 ,ul ofthe apl)lol,l;ate termination mixture.

Termination mixtures were made in Klenow buffer and comprise the nucleotide concentrations shown below in Table 11.
Table 11 Termin~tion dATP dGTP dCTP dTTP ddNTPs Mix inmM in mM inmM inmM
ddATP mix 10 100 100 100100 mM ddATP
ddGTP mix 100 5 100 100120 mM ddGTP
ddCTP mix 100 100 10 100100 mM ddCTP
ddTTP mix 100 100 100 5500 mM ddTTP
Termination mixtures were incubated for 20 minutes at ambient tempe~ e. Two ~11 of chase solution (0.5 mM of each of four dNTPs in Klenow buffer) were added to each reaction tube and mixtures were incubated for another 15 minutes, again at ambient temperature.
15 Magnetic beads were precipitated with a magnetic particle concentrator (or centrifugation) and the supernatant discarded. Beads were resuspended in a solution containing 10 ~1 of deionized forrnamide, 5 mg/ml dextran blue and 0.1% SDS, and heated to 95~C for 5 minutçs, and stored on ice for less than 10 minlltçs. Samples were analyzed on a DNA sequencing gel and on 20 an ALF DNA sequencer (Pharmacia; Piscataway, NJ) using a 6%
polyacrylamide gel with 7 M urea and 0.6 x TBE. Surprisingly, sequencing reactions performed in the presence of single-stranded DNA binding protein showed considerable improvement in resolution. Only 50 bases were resolved from reactions performed without single-stranded DNA binding 25 protein (Figure 18, bottom panel) whereas 200 bases could be resolved from WO 96/32!j04 PCI/US96/05136 re~r,ti~n~ ~)tl rO- ..,e-1 iIl the presence of single-stranded DNA binding prol~(Figure 18, top panel).
Exarnple 23 Specificity of Double-Strand Se~llencin~ by Strand Displacement.
S Another experiment was performed to delf . .. ,i~,e the specificity and applicability of the nick translation strand displacement method of sequencing double-stranded nucleic acids. A schem~tic of the experimPnt~l design is shown in Figure 19. Briefly, a double-stranded target DNA was ~l~pared by digesting double-stranded q?X174 phage DNA with 0 TspR I restriction en~lonllclease. TspR I has a recognition site of NNCAGTGNN and cleaves q~X174 into 12 fr~ment~ each with distinctive 3' protruding ends. Possible ends are shown in Table 12.
Table 12 5'-AACACTGAC-3' 7 5'-GTCAGTGTT-3' 2 5'-AACAGTGGA-3' 8 5'-GTCAGTGGT-3' 3 5'-ACCACTGAC-3' 9 5'-GTCACTGAT-3' 4 5'-AACACTGGT-3' 10 5'-TCCACTGTT-3' 5'-ATCAGTGAC-3' 11 5'-TGCAGTGGA-3' 6 5'-ACCAGTGTT-3' 12 5'-TCCACTGCA-3' q~X174 DNA (5 pmol) was dephosphorylated using calf inteshn~l aLkaline phosphatase. Briefly, q~X174 DNA was resuspended in 100 ,ul buffer (500 rnM Tris-HCl, pH 9.0, 1 mM MgCl2, 0.1 mM ZnCl2, and 1 mM sperrni~line) cu~ 5 units of calf intestinal aLkaline phosphatase 25 (Promega; Madison, WI). The reaction was incubation at 37~C for 15 es and at 56~C for 15 minutes. Five additional units of calf intestinal SUB~ ITE SHEET ~RULE 26) ~lk~line ph~ ~ph~t~e was added and a second incubation was performed at 37~C for lS minlltes and at 56~C for 15 minl-tçs DNA in the samples was extracted once with phenol, once with phenol/chloroform, and once with chloroform, after which nucleic acid was precipitated in 0.3 M sodium 5 acetate/2.5 volumes ethanol. Precipitated ~X174 DNA was washed twice with TE and resuspended in 300 ,ul of IE cont~inin~ 1 M NaCl.
Double-stranded probes, comprising biotin (B), fluorescein (F), and infra dye (CY5) labels, were synthesized and anchored to magnetic beads as shown in Table 13.

WO 96/325~4 PCTIU~9C1~5136 Table 13 -DF27-1 5~-GATGATCCGACGCATCACATCAGTGAC-3' (SEQ ID NO. 34) 3~-CTACTAGGCTGCGTAGTG-p-5' (SEQID NO.35) DF27-2 5'F-GATGATCCGACGCATCACTCCACTGTT-3' (SEQ ID NO.36) 3~-CTACTAGGCTGCGTAGTG-p-5' (SEQ ID NO. 37) DF27-3 5~-GATGATCCGACGCATCACGTCAGTGTT-3' (SEQ ID NO. 38) 3~-CTACTAGGCTGCGTAGTG-p-5' (SEQ ID NO. 39) DF27-4 5~-GATGATCCGACGCATCACTGCAGTGGA-3' (S~Q ID NO. 40) 3~-CTACTAGGCTGCGTAGTG-p-5' (SEQ ID NO. 41) DF27-5-CY5 5'CY5-GATGATCCGACGCATCACGTCACTGAT-3' (SEQ ID NO. 42) 3~-CTACTAGGCTGCGTAGTG-p-5' (SEQ ID NO. 43) DF27-6-CY5 S'CY5-GATGATCCGACGCATCACAACAGTGGA-3' (SEQ ID NO. 44) 3'B-CTACTAGGCTGCGTAGTG-p-5' (SEQ ID NO. 45) DF27-7 5'-F-GATGATCCGACGCATCACGTCAGTG~T-3' (SEQ ~ NO. 46) 3~-CTACTAGGCTGCGTAGTC-p-5' (SE~Q ID NO. 47) DF27-8 5'-F-GATGATCCGACGCATCACAACACTGGT-3' (SEQ ID NO. 48) 3'B-CTACTAGGCTGCGTAGTG-p-S' (SEQ ID NO. 49) 0 DF27-9 5'-F-GATCATCCCAGGGATCACAAGAGTGAC-3' (SEQ ID NO. 50) 3'B-CTACTAGGGTCCCTAGTG-p-5' (SEQ ID NO.51) DF27-10 5'-F-GATGATCCGACGCATCACACCACTGAC-3' (SEQID NO.52) 3~-CTACTAGGCTGCGTAGTG-p-S' (SEQID NO.53) Beads with about 25 pmol of immobili7~d primer were ligated to 3 pmol of digested TspR I ~Xl 74 DNA in 50 ~11 co~ g 400 units of 15 T4 DNA ligase (New Fngl~n~l Biolabs; Beverly, MA), 50 mM Tris-HCl, pH
7.8, 10 mM MgC12, 10 mM dithio~eilol, 1 mM ATP and 25 ~Lg/ml bovine serum albumin. Ligation reactions were performed at 37~C for 30 mimltes, at 50~C to 55~C for one hour (thermal ligase), at room temperature for 2 SlJB~ E SHEET (F~ULE 26) WO 96132504 PCI~/US96/OS136 hours or at 4~C for overni~ht After ligation, beads were washed twice with TE and resuspended in 300 ,ul ofthe same buffer.
SeqllPncin~ reactions: For each sequencing reaction, 30 ,ul of beads cv. ,~ the ligation product was used. Beads were resuspended in 5 a 13 ,ul volume co~ 1.5 ,ul of 10 x Klenow buffer (100 mM Tris-HCl, pH 7.5, 50 mM MgCl2 and 75 mM dithiothreitol), and with or without 1 ~11 of single-stranded DNA binding protein (SSB, 5 ,ug/~ll; USB; Cleveland, Ohio). Reaction ~ cs were incubated on ice for S mimltes, followed by the addition of 5 units of Klenow Fr~ ment (New Fngl~nd Biolabs). The reaction volume was split into four termin~tion mixes, each consisting of 1 ~11 DMSO plus 3 ~Ll ofthe al)pr~liate t~rmin~tion mix. Termin~tion mixes were made in Klenow buffer and comprise the nucleotides concentrations shown in Table 11.
Termin~tion mixtures were incubated for 20 minutes at ambient temperature. Two ~11 of a chase solution cont~ining 0.5 mM of each of the four dNTPs in Klenow buffer, was added to each reaction tube and mixtures were incubated for another 15 minntes at ambient temperature.
Beads were precipitated by magnetic particle concentrator or centrifugation and the supern~t~nt discarded. Precipitated beads were resuspended in TE
or in a solution cO,~t~ 10 ,ul deionized form~mi-1e, 5 mg/ml dextran blue and 0.1% SDS, and heated to 95~C for 5 minlltes Mixtures were stored on ice for less than 10 min~tes and analyzed by a DNA sequencing gel and on an ALF DNA sequencer (Pharmacia; Piscataway, NJ) using a 6%
polyacrylamide gel with 7 M urea and 0.6 x TBE.
One double stranded primer was used for each reaction and the results achieved using primers DF27- 1, DF27-2, DF27-4, DF27-5-CY5 and .~ CA 02218188 1997-10-14 WO Sf~2''01 1 ~iu~ 5 lQ3 DF27-6-CY5, are shown in Figures 20, 21, 22, 23 and 24, respectively.
Each prirner was capable of ~n~ratin~ sequencing inf~ ion of up to 200 b~eep~;rs without significant interference from the 11 f~agments with non-compk;.--F............ ........~ , ende.
Other embo.iim~ontc and uses ofthe invention will be a~a.c.lt to those skilled in the art from consideration ofthe specification and practice of the invention disclosed herein. All U.S. Patents and other references noted herein are specifically incorporated by reference. The specification and examples should be coneitlered exemplary only wi~ the true scope and spirit of the invention indicated by the following claims.

Claims (91)

We Claim:

1. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of said target;
b) hybridizing said set to an array of nucleic acid probes, wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, to form a target array of nucleic acids, c) determining molecular weights for a plurality of nucleic acids of said target array; and d) determining the sequence of said target nucleic acid.

2. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acid fragments each containing a sequence that corresponds to a sequence of said target;
b) hybridizing said set to an array of nucleic acid probes, wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion;
c) creating a mass modified extended nucleic acid by extending and mass modifying a strand of the probe using the hybridized fragment as a template;
d) determining molecular weights for a plurality of mass modified extended nucleic acids by mass spectrometry; and e) determining the sequence of said target nucleic acid.

3. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;
b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, and for each complex;
i) ligating a single strand of the fragment to an adjacent single strand of the probe; and ii) extending the unligated strand of the complex by strand-displacement polymerization using the ligated strand as a template; and c) determining the sequence of the target.

4. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acid fragments each containing a sequence which corresponds to a sequence of said target;
b) hybridizing said set of fragments to an array of mass modified probes, wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion;

c) extending a strand of the mass modified probes using the hybridized fragments as templates;
d) determining molecular weights for a plurality of extended mass modified strands; and e) determining the sequence of said target.

5. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;
b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes, and for each complex, i) ligating a single strand of the fragment to an adjacent single strand of the probe; and ii) extending the unligated strand of the complex by strand-displacement polymerization using the ligated strand as a template and mass-modifying the extended strand;
c) determining the molecular weights of the extended strands by mass spectrometry; and d) determining the sequence of the target from the molecular weights of the extended strands.

6. A method for sequencing a target nucleic acid comprising the steps of:

a) providing a set of nucleic acids complementary to a sequence of said target;
b) hybridizing said set to an array of single-stranded nucleic acid probes wherein each probe comprises a constant sequence and a variable sequence and said variable sequence is determinable;
c) determining molecular weights of hybridized nucleic acids;
and d) identifying the sequence of said target.

7. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acids homologous to a sequence of said target;
b) hybridizing said set to an array of single-stranded nucleic acid probes wherein each probe comprises a constant sequence and a variable sequence;
c) determining molecular weights of hybridized nucleic acids;
and d) identifying the sequence of said target.

8. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of partially single-stranded nucleic acid fragments wherein each fragment contains a sequence that corresponds to a sequence of the target;
b) hybridizing the single-stranded portions of the fragments to single-stranded portions of a set of partially double-stranded nucleic acid probes to form a set of complexes wherein each probe contains a variable sequence within the single-stranded region, and for each complex;
i) ligating a single strand of the fragment to an adjacent single strand of the probe; and ii) extending the unligated strand of the complex by strand displacement polymerization using the ligated strand as a template, c) determining the molecular weights of the extended strands by mass spectrometry, and d) determining the sequence of the target from the molecular weights of the extended strands.

9. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acid fragments each containing a sequence which corresponds to a sequence of said target;
b) hybridizing said set to an array of nucleic acid probes, wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion;
c) extending a strand of the probe enzymatically using the hybridized fragment as a template to create an extended nucleic acid;
d) removing alkali cations from said extended nucleic acid;
e) determining molecular weights for a plurality of protonated and extended nucleic acids by mass spectrometry; and f) determining the sequence of said target.

10. A method for sequencing a target nucleic acid comprising the steps of:
a) providing a set of nucleic acid fragments each containing a sequence which corresponds to a sequence of said target;
b) hybridizing said set to an array of nucleic acid probes wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, to form a target array of nucleic acids;
c) extending a strand of the probe using the hybridized fragment as a template;
d) determining molecular weights for a plurality of nucleic acids of said target array; and e) determining the sequence of said target.

11. A method for sequencing a target nucleic acid comprising the steps of:
a) fragmenting a sequence of the target into nucleic acid fragments;
b) hybridizing said fragments to an array of nucleic acid probes wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion and said array is attached to a solid support;
c) determining molecular weights of hybridized fragments by mass spectrometry;

d) determining nucleotide sequences of the hybridized fragments; and e) identifying the sequence of said target.

12. The method of claims 1-11 wherein the target nucleic acid is obtained from a biological or recombinant source.

13 . The method of claims 1-11 wherein the target nucleic acid and the probe are each between about 10 to about 1,000 nucleotides in length.

14. The method of claims 1-11 wherein the sequence is homologous with at least a portion of said target sequence.

15. The method of claims 1-11 wherein the sequence is complementary to at least a portion of said target sequence.

16. The method of claims 1-11 wherein the set, the fragments or the probes are dephosphorylated by treatment with a phosphatase prior to hybridization.

17. The method of claims 1-11 wherein the set or the fragments are created by enzymatically or physically cleaving said target, or by enzymatically replicating said target with chain terminating and chain elongating nucleotides.

18. The method of claims 1-5 or 8-1 1 wherein the fragments comprise a nested set.

19. The method of claims 1-11 wherein the target, the fragments and the probes comprise DNA, RNA, PNA or modifications or combinations thereof.

20. The method of claims 1-11 wherein the fragments are provided by synthesizing a complementary copy of the target sequence and fragmenting said target sequence by nuclease digestion.

21. The method of claims 1-11 wherein the fragments are provided by enzymatically polymerizing complementary copies of said target with chain terminating and chain elongating nucleotides.

22. The method of claims 1-11 wherein the nucleic acid fragments comprise greater than about 10 4 different members and each member is between about 10 to about 1,000 nucleotides in length.

23. The method of claims 1-11 wherein the set or the target fragments is provided by enzymatically polymerizing complementary copies of said target with chain terminating and chain elongating nucleotides.

24. The method of claim 23 wherein enzymatic polymerization is a nucleic acid amplification process selected from the group consisting of strand displacement amplification, ligase chain reaction, Q.beta. replicase amplification, 3SR amplification and polymerase chain reaction amplification.

25. The method of claims 6 or 7 wherein the constant sequence is between about 3 to about 18 nucleotides in length.

26. The method of claims 1-11 wherein the single-stranded portion of each probe contains a variable sequence of between about 4 to about 9 nucleotides in length.

27. The method of claims 1-11 wherein the fragments, the set of nucleic acids or the probes are attached to a solid support.

28. The method of claims 1 - 11 wherein each probe is between about 10 to about 50 nucleotides in length.

29. The method of claims 1-5 or 8-11 wherein the double-stranded regions of the probes contain the same sequence for each probe of the set.

30. The method of claims 1-11 further comprising the step of ligating hybridized fragments to said probes.

31. The method of claims 1-11 further comprising the step of extending a strand of the probe using the hybridized fragment as a template wherein the extended strand displaces the hybridized fragment.

32. The method of claim 31 wherein the extended strand comprises between about 0.1 femtomole to about 1.0 nanomole of nucleic acid.

33. The method of claim 31 wherein the extended strand is between about 10 to about 100 nucleotides in length.

34. The method of claims 1 - 11 wherein there are less than or equal to 4R
different probes and R is the length in nucleotides of the variable sequence.

35. The method of claim 27 wherein the solid support is selected from the group consisting of plates, beads, microbeads, whiskers, combs, hybridization chips, membranes, single crystals, ceramics and self-assembling monolayers.

36. The method of claim 27 wherein the probes are conjugated with biotin or a biotin derivative and the solid support is conjugated with avidin, streptavidin or a derivative thereof.

37. The method of claim 27 wherein the probes are attached to said solid support by covalent bond, an electrostatic bond, a hydrogen bond, a photocleavable bond, an electrostatic bond, a disulfide bond, a peptide bond, a diester bond, a selectively releasable bond or a combination thereof.

38. The method of claim 37 wherein the attachment is a cleavable attachment which is cleavable by heat, an enzyme, a chemical agent or electromagnetic radiation.

39. The method of claim 38 wherein the chemical agent is selected from the group consisting of reducing agents, oxidizing agents, hydrolyzing agents and combinations thereof.

40. The method of claim 38 wherein the electromagnetic radiation is selected from the group consisting of visible, ultraviolet and infrared radiation.

41. The method of claim 37 wherein the selectively releasable bond is 4,4'-dimethoxytrityl or a derivative thereof.

42. The method of claim 41 wherein the derivative is selected from the group consisting of 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-methyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-hydroxymethyl-benzoic acid, N-succinimidyl- 3 or 4 [bis-(4-methoxyphenyl)]-chloromethyl-benzoic acid and salts thereof.

43. The method of claim 27 further comprising a spacer between the probe and the solid support.

44. The method of claim 43 wherein the spacer is selected from the group consisting of oligopeptides, oligonucleotides, oligopolyamides, oligoethyleneglycerol, oligoacrylamides, alkyl chains of between about 6 to about 20 carbon atoms and combinations thereof.

45. The method of claims 1, 6, 7 or 11 wherein the probe is extended using the hybridized strand as a template.

46. The method of claims 2-5, 8-10 or 45 wherein extending comprise polymerization incorporating mass-modifying nucleotides into the extended strand.

47. The method of claims 2-5, 8-10 or 45 wherein the strand is extended enzymatically using chain terminating and chain elongating nucleotides.

48. The method of claims 2-5, 8-10 or 45 wherein a plurality of extended strands comprise about 0.1 femtomole to about 1.0 nanomole of nucleic acid.

49. The method of claims 1-5 or 8-11 wherein the sequence is determined by polyacrylamide electrophoresis, capillary electrophoresis or mass spectrometry.

50. The method of claim 46 wherein the mass modified extended nucleic acid comprises between about 0.1 femtomole to about 1.0 nanomole of nucleic acid.

51. The method of claim 46 wherein the mass modified extended nucleic acid is between about 10 to about 100 nucleotides in length.

52. The method of claim 46 wherein the mass modified extended strand contains a plurality of mass modifying functionalities.

53. The method of claims 1-11 wherein the strand of said probe is mass modified by enzymatically extending said strand using a polymerase and a mass modified nucleotide.

54. The method of claim 53 wherein the mass modified nucleotide is a chain elongating or chain terminating nucleotide.

55. The method of claim 53 wherein the mass modified nucleotide contains a plurality of mass modifying functionalities;

56. The method of claim 53 wherein the mass modified probes contain a plurality of mass modifying functionalities.

57. The method of claims 52, 55 or 56 wherein at least one mass modifying functionality is coupled to a heterocyclic base, a sugar moiety or a phosphate group.

58. The method of claims 52, 55 or 56 wherein the mass modifying functionality is a chemical moiety that does not interfere with hydrogen bonding for base-pair formation.

59. The method of claims 52, 55 or 56 wherein the mass modifying functionality is coupled to a purine at position C2, N3, N7 or C8 or a deazapurine at position N7 or C9.

60. The method of claims 52, 55 or 56 wherein the mass modifying functionality is coupled to a pyrimidine at position C5 or C6.

61. The method of claims 52, 55 or 56 wherein the mass modifying functionality is selected from the group consisting of deuterium, F, Cl, Br, I, SiR, Si(CH3)3, Si(CH3)2(C2H5), Si(CH3)2(C2H)5,2 Si(CH )(3C H2) 6 2 Si(C2H5)3, (CH2)nCH3, (CH2)aNR, CH2CONR, (CH2)nOH, CH2F, CHF2 and CF3; wherein n is an integer and R is selected from the group consisting of -H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

62. The method of claims 52, 55 or 56 wherein the mass modifying functionality is generated from a precursor functionality which is -N3 or-XR, wherein X is selected from the group consisting of -OH, -NH2, -NHR,-SH, -NCS, -OCO(CH2)nCOOH, -NHCO(CH2)nCOOH, -OSO2OH, -OCO(CH2)nI and -OP(O-alkyl)-N-(alkyl)2, and n is an integer from 1 to 20;
and R is selected from the group consisting of-H, deuterium and alkyls, alkoxys and aryls of 1-6 carbon atoms, polyoxymethylene, monoalkylated polyoxymethylene, polyethylene imine, polyamide, polyester, alkylated silyl, heterooligo/polyaminoacid and polyethylene glycol.

63. The method of claims 1, 6, 7 or 11 wherein the hybridized nucleic acid fragment is extended.

64. The method of claims 2, 3, 4, 5, 8-10 or 63 wherein the extended nucleic acid is mass modified by thiolation.

65. The method of claim 64 wherein thiolation is performed by treating said extended strand with a Beaucage reagent.

66. The method of claims 2, 3, 4, 5, 8-10 or 63 wherein the extended nucleic acid is mass modified by alkylation.

67. The method of claim 66 wherein alkylation is performed by treating said extended strand with iodoacetamide.

68. The method of claim 66 further comprising the step of removing alkali cations from said mass modified extended nucleic acid.

69. The method of claim 68 wherein alkali cations are removed by ion exchange.

70. The method of claim 69 wherein ion exchange comprises contacting said extended nucleic acid with a solution selected from the group consisting of ammonium acetate, ammonium carbonate, diammonium hydrogen citrate, ammonium tartrate and combinations thereof.

71. The method of claims 2, 5, 8, 9, 11 or 49 wherein mass spectrometry includes a release step selected from the group consisting of laser heating, droplet release, electrical release, photochemical release and electrospray.

72. The method of claims 2, 5, 8, 9, 11 or 49 wherein mass spectrometry includes an analytical step selected from the group consisting of Fourier Transform, ion cyclotron resonance, time of flight analysis with reflection, time of flight analysis without reflection and quadrupole analysis.

73 . The method of claims 2, 5, 8, 9, 11 or 49 wherein mass spectrometry is performed by fast atom bombardment, plasma desorption, matrix-assisted laser desorption/ionization, electrospray, photochemical release, electrical release, droplet release, resonance ionization or a combination thereof.

74. The method of claims 2, 5, 8, 9, 11 or 49 wherein mass spectrometry includes time of flight with reflection, time of flight without reflection, electrospray, Fourier transform, ion trap, resonance ionization, ion cyclotron resonance or a combination thereof.

75. The method of claims 1, 2, 4, 6, 7, 9, 10 or 11 wherein two or more molecular weights are determined simultaneously.

76. The method of claims 1, 2, 4, 6, 7, 9, 10 or 11 wherein molecular weights are determined by matrix-assisted laser desorption ionization mass spectrometry and time of flight analysis.

77. The method of claims 1, 2, 4, 6, 7, 9, 10 or 11 wherein molecular weights are determined by electrospray ionization mass spectrometry and quadrupole analysis.

78. A method for detecting a target nucleic acid comprising the steps of:
a) providing a set of nucleic acids complementary to a sequence of said target;

b) hybridizing said set to a fixed array of nucleic acid probes wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion which is determinable;
c) determining molecular weights of hybridized nucleic acids by mass spectrometry; and d) identifying a sequence of the target.

79. A method for detecting a target nucleic acid comprising the steps of:
a) providing a set of nucleic acids complementary to a sequence of said target;
b) hybridizing said set to a fixed array of nucleic acid probes wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion to form a target array of nucleic acids;
c) mass modifying a plurality of nucleic acids of said target array;
d) determining molecular weights of the mass modified nucleic acids by mass spectrometry; and e) identifying a sequence of the target.

80. The method of claims 78 or 79 wherein the target is provided from a biological sample.

81. The method of claim 80 wherein the sample is obtained from a patient.

82. The method of claims 78 or 79 wherein detection of the target is indicative of a disorder in the patient.

83. The method of claims 78 or 79 wherein the disorder is a genetic defect, a neoplasm or an infection.

84. An array of nucleic acid probes wherein each probe comprises a first strand and a second strand wherein said first strand is hybridized to said second strand forming a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, and said array is attached to a solid support comprising a material that facilitates volatization of nucleic acids for mass spectrometry.

85. An array of single-stranded nucleic acid probes wherein each probe comprises a constant sequence and a variable sequence which is determinable, and said array is attached to a solid support comprising a matrix that facilitates volatization of nucleic acids for mass spectrometry.

86. The array of claims 84 or 85 wherein the nucleic acid probes are mass modified nucleic acid probes.

87. The array of claims 84 or 85 which contains less than or equal to about 4R different probes and R is the length in nucleotides of the variable sequence.

88. A kit for detecting a sequence of a target nucleic acid comprising an array of nucleic acid probes fixed to a solid support wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, and the solid support comprises a matrix chemical that facilitates volatization of nucleic acids for mass spectrometry.

89. A kit for detecting a sequence of a target nucleic acid comprising an array of mass modified nucleic acid probes fixed to a solid support wherein each probe comprises a double-stranded portion, a single-stranded portion and a variable sequence within said single-stranded portion, and the solid support comprises a matrix chemical that facilitates volatization of nucleic acids for mass spectrometry.

90, for determining sequence information comprising a mass spectrometer, a computer and an array of mass modified nucleic acid probes wherein each probe comprises a single-stranded portion, an optional double-stranded portion and a variable sequence within said single-stranded portion, and wherein said array is attached to a solid support.

91. for determining sequence information comprising a mass spectrometer, a computer and an array of nucleic acid probes wherein each probe comprises a single-stranded portion, an optional double-stranded portion and a variable sequence within said single-stranded portion, and wherein said array is attached to a solid support.