MXPA98002007A - Cebers modify - Google Patents

Abstract

The present invention relates to modified oligonucleotides for use in the amplification of a sequence of nucleic acids. The amplifications carried out using the modified oligonucleotides lead to a less specific non-specific amplification product, in particular, the primer-dimer, and to a concomitant greater yield of the proposed amplification product compared to the amplifications carried out using the oligonucleotides not modified as the primers

Description

MODIFIED PRIMERS Field of the Invention The present invention relates to the field of molecular biology and the chemistry of nucleic acids. More specifically, it relates to methods and reagents for improving the field of nucleic acid amplification reactions. The invention, therefore, has applications in any field in which nucleic acid amplification is used.

Background of the Invention The invention of the polymer chain reaction (PCR) made possible the in vitro amplification of the nucleic acid sequences. PCR is described in U.S. Pat. Nos. 4,683,195; 4,683,202; and 4,965,188; Saiki et al., 1985, Science 230: 1350-1354; Mullis et al., 1986, Cold Springs Harbor Symp. Quant. Biol. 51: 263-273; and Mullis and Faloona, 1987, Methods Enzymol. 155: 335-350. The development and application of PCR are described extensively in the literature. For example, a range of issues related to PCR Ref.027043 are described in PCR Technology - Principles and Applications for DNA Amplification, 1989, (ed. H.A. Erlich) Stockton Press, New York; PCR Protocols: A guide to methods and applications, 1990, (ed M.A. Innis et al) Academic Press, San Diego; and PCR Strategies, 1995, (ed. M.A. Innis et al.) Academic Press, San Diego. Commercial vendors, such as Perkin Elmer (Norwalk, CT), commercialize PCR reagents and publish PCR protocols. Since the original publication of nucleic acid amplification, several primer-based nucleic acid amplification methods have been described including, but not limited to, the Ligase Chain Reaction (LCR, Wu and Wallace, 1989, Genomic 4: 560-569 and Barany, 1991, Proc. Nati, Acad. Sci. USA 88 ^: 189-193); the Ligase Polymerase Chain Reaction (Barany, 1991, PCT Methods and Applic 1: 5-16); Gap-LCR (PCT patent publication No. WO 90/01069); Reaction in Repair Chain (publication of European Patent Application No. 439,182 A2), 3SR (Kwoh et al., 1989, Proc. Nati. Acad. Sci. USA 86: 1173-1177, Gautelli et al., 1990 , Proc. Nati, Acad. Sci. USA T7: 1874-1878, publication of PCT patent application No. WO 92 / 0880A), and NASBA (US Patent No. 5,130,238). An examination or analysis of the systems of ? 'Amplification is provided in Abramson and Miers, 1993, Current Opinion in Biotechnology 4: 41-47. The specify of the amplification reactions based on a primer depends on the specify of primer hybridization. Under the typical high amplification temperatures used, the primers hybridize only to the target sequence or target. However, the amplification reaction mixtures are coupled typically at room temperature, well below the temperature necessary to ensure the specify of the primer hybridization. Under such less stringent conditions, the primers can bind non-specifically to other nucleic acid sequences only partially Complementary or other primers and initiate the synthesis of the undesirable extension products, which j? T can be amplified in the company of the target or target sequence. The amplification of non-specific primer extension products can compete with the amplification of the desired target or target sequences and can significantly reduce the effncy of the amplification of the desired sequence. A frequently observed type of non-specific amplification product is an artifact independent of the matrix or model of the amplification reactions referred to as the "primer-dimer". He primer-d is a double-stranded fragment whose length is typically close to the sum of the two primer lengths and seems to occur when one primer is spread over the other primer. The resulting concatenation or chaining forms an undesirable model or matrix which, because of its short length, it is amplified efficiently. The non-specific amplification can be reduced by decreasing the formation of the extension products of the primer prior to the start of the reaction. In one method, referred to as a "hot start" protocol, one or more critical reagents are retained from the reaction mixture until the temperature is raised to a level sufficient to provide the specificity of the necessary hybridization. In this way, the reaction mixture can not withstand the extension of the primer for the period of time that the reaction conditions do not ensure hybridization of the specific primer. Hot-start, manual methods, in which the reaction tubes are open after the incubation step at elevated temperature and disappearance reagents are added, are labor intensive and increase the risk of contamination of the reaction mixture. Alternatively, a heat sensitive material, such as wax, can be used to separate sequestering or separate reaction components, as described in U.S. Pat. No. 5,411,876, and Chou et al., 1992, Nucí. Acids Res. 20 (7): 1717-1723. In these methods, an incubation of the pre-reaction at high temperature melts the heat-sensitive material, whereby the reactants are allowed to mix. Another method to reduce the formation of the primer extension products prior to the start or start of the reaction lies in the reversible heat inhibition of the DNA polymerase by the specific antibodies to the DNA polymerase, as described in U.S. Pat. No. 5,338,671. The antibodies are incubated with the DNA polymerase in a buffer solution at room temperature prior to assembly of the reaction mixture to allow the formation of the polymerase complex of the DNA-antibody. The inhibition of the antibodies of the DNA polymerase activity is inactivated by an incubation of the pre-reaction at elevated temperature. A disadvantage of this method is that the production of specific antibodies to the DNA polymerase is expensive and time-consuming, especially in large quantities. In addition, the addition of antibodies to a reaction mixture may require redesigning the amplification reaction.

The formation of extension products prior to the start of the reaction can also be inhibited by the addition to the reaction of a single-stranded binding protein, which binds non-covalently to the primers in a heat-reversible manner and inhibits the extension of the primer preventing or preventing hybridization. The non-specific amplification can also be reduced by enzymatically degrading the extension products formed prior to the start of the reaction using the methods described in U.S. Pat. No. 5,418,149. The degradation of the recently synthesized extension products is achieved by the incorporation into the reaction mixture of dUTP and UNG, and incubating the reaction mixture at 45-60 ° C prior to carrying out the amplification reaction. The extension of the primer leads to the formation of the uracil-containing DNA, which is degraded by the UNG under the pre-amplification conditions. A disadvantage of this method is that the degradation of the extension product is due to the formation of the extension product and the elimination of the product from the extension of the non-specific primer is likely to be less complete. An advantage of this method is that the DNA containing uracil, introduced into the reaction mixture as a contamination from a previous reaction is also degraded and, therefore, the method also reduces the problem of contamination of a PCR by the amplified nucleic acid prior to the previous reactions. The conventional techniques of molecular biology and chemistry of nucleic acids, which are within the experience of the technique, are fully explained in the literature. See, for example, Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor, Laboratory, Cold Spring Harbor, New York; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Acid Hybridization (B. D. Ha and S. Higgins, eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.). The present invention provides covalently modified oligonucleotide primers for the in vitro amplification of nucleic acid sequences. The use of the modified primers of the invention leads to a reduction in non-specific amplification, especially primer-dimer formation, and / or a concomitant increase in the proposed target field relative to an amplification carried out with non-specific primers. modified. In one aspect the invention relates to an oligonucleotide primer for the amplification of a nucleic acid sequence, having the general structure: R R I I S'-S-L- -S 'or 5'-S? -N-S -3 wherein Si represents a first nucleotide sequence between about 5 and about 50 5 nucleotides in length; wherein S2 represents a second sequence between one and three nucleotides in length; where N represents a nucleotide that is either fl | it contains a purine or a pyrimidine base containing an exocyclic amine; wherein R represents a modifying group, wherein R is covalently bonded to N through the exocyclic amine, and wherein R has the structure: 0 wherein R ± and R 2 independently represent hydrogen, an alkyl group with C 1 -C 0, an alkoxy group, a phenyl group, a phenoxy group, a phenyl group substituted, a naphthyl group, or a substituted naphthyl group. The alkyl groups can be branched or unbranched. In a preferred embodiment, N is a modified conventional nucleotide, in which case N is an adenosine, Cytidine, or modified guanosine, and the modifying portion is covalently attached to the amine Exocyclic base of adenine, guanine, or cytosine. In a more preferred embodiment, N is a modified adenosine. In a preferred embodiment, R is a 2-naphthylmethyl group; a benzyl group; or a substituted benzyl group. Preferred substituted benzyl groups have the structure: ^ O where R3 represents a linear or branched alkyl group with Ci-Cβ more preferably a linear or branched alkyl group with C? -C, a methoxy group, or a nitro group. Preferably, R3 is fixed in the para position. In a more preferred embodiment, R is a benzyl, p-methylbenzyl, p-tert-butylbenzyl, p-mctoxybenzyl, or 2-naphthylmctyl group. Another aspect of the invention relates to amplification primers which are modified by a photolabile binding or covalent attachment of a modifying group, which leads to a partial or complete inhibition of the extension of the primer. The photo-labile modifier can be linked either to the exocyclic amine, or to the modified nucleotides described JJP above, or to the ring nitrogen. In one embodiment, at least one nitrobenzyl group is attached to the exocyclic amine of an adenine, guanine, or cytosine base of the 3 'terminal nucleotide. Another aspect of the invention is a pair or set of primers, wherein at least one of the primers is modified as described above. In a preferred embodiment, both elements of a pair of primers or all members of a set of primers, are modified. Another aspect of the invention relates to methods for amplifying the nucleic acid, which comprise carrying out an amplification reaction using the modified primers of the invention. Another aspect of the invention relates to methods for exemplifying a target or target nucleic acid which comprises carrying out an amplification reaction using the photolabile modified primers of the invention, wherein the mixture of the The reaction is irradiated with sufficient light to remove the modifier group and to allow the formation of the primer extension products. In one embodiment of the invention, the irradiation is carried out as a separate step, prior to the start or start of the reaction of amplification, but after the reaction mixture has been heated to a higher temperature than approximately 50 ° C. In other embodiments, the irradiation step is combined with a preliminary step of the amplification process, such as the step of reverse transcription of an RNA amplification reaction, or the initial denaturation step in a DNA amplification reaction. . Another aspect of the invention relates to sets or sets for the in vitro amplification of nucleic acid sequences, such sets or sets comprise a pair of primers in which at least one of the primers is modified as described herein. The kits or kits of the present invention also include one or more amplification reagents, for example, a polymerase or nucleic acid ligimerase, nucleoside triphosphate, and suitable buffer solutions.

Brief Description of the Drawings Figure 1 shows the results of the HIV-1 RNA amplifications carried out using the benzylated primers, as described in Example 5. Figure 2 shows the results of the VCH RNA amplifications carried out using the benzylated primers, as described in Example 6. Figure 3 shows the result of VCH RNA amplifications carried out using the modified primers with one of three modifier groups, as described in Example 7. Figure 4 shows the results of the VCH RNA amplifications carried out using photolabile modified primers, as described in Example 8. Figure 5 shows the results of the amplifications of the mycobacterial DNA using the modified primers with a benzyl group and modified primers with a p-tert-butylbenzyl group, as described in Example 10. Figure 6 shows a general reaction scheme suitable for the synthesis of modified pore size glass (CPG) modified with dA modified with benzyl or substituted benzyl. To help understand the invention, several terms are defined immediately. The terms "nucleic acid" and "oligonucleotide" refers to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), to polyribonucleotides (containing D-ribose), and to any other type of polynucleotide which is an N glycoside of a purine base or pyrimidine, or a modified pyrimidine or purine base. There is no proposed distinction in length between the terms "nucleic acid" and "oligonucleotide", and these terms will be used interchangeably. The terms refer only to the primary structure of the molecule. Accordingly, these terms include single-stranded or double-stranded DNA, as well as single-stranded or double-stranded RNA. The term "conventional", with reference to the bases of nucleic acids, nucleosides, or nucleotides, refers to those which are naturally present in the polynucleotide that is described. The four conventional DNA deoxyribonucleotides (also referred to as major) contain the adenine and guanine of the purine bases and the cytosine and thymine of the pyrimidine bases. The four conventional RNA ribonucleotides contain the adenine and guanine of the purine bases and the cytosine and uracil of the pyrimidine bases. In addition to the conventional or common bases above, several other purine and pyrimidine derivatives, called the rare or minor bases, are present in small amounts in some nucleic acids. When used here, "unconventional," with reference to the bases of nucleic acids, nucleosides, or nucleotides, refers to nucleic acid bases rare or minor, nucleosides, or nucleotides, and modifications, derivations, or analogs of the bases, nucleosides, or conventional nucleotides, and include synthetic nucleotides having modified base portions and / or modified sugar portions (see, Protocols for Oligonucleotide Conjugates , Methods in Molecular Biology, Vol. 26, (Sudhir Agrawal, Ed., Humana Press, Totowa, NJ, (1994)) and Oligonucleotides and Analogues, A practical Approach (Fritz Eckstein, Ed., IRL Press, Oxford University Press , Oxford.) Oligonucleotides can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a tai method such as the phosphate triester method of Narang et al., 1979, Meth. Enzymol 68_: 90-99, the phosphors dicster method of Brown et al., 1979, Meth. Enzymol 68: 109-151, the diethyl phosphoramidite method of Beaucage et al., 1981, Tetrahedro n Lett.22: 1859-1862, and the solid support method of U.S. Pat. No. 4,458,066. A review of synthesis methods is provided in Goodchild, 1990, Bioconjugate Chemistry 1. (3): 165-187, incorporated herein by reference. The term "base parking", also referred to in the art as "Watson-Crick base mating", refers to the hydrogen bonding well of the complementary base pairs of adenine-ti ina and guanine-cytosine in a structure of double-stranded DNA, adenine-uracil and guanine-cytosine in a hybrid RNA / DNA molecule, and to the ogous binding of unconventional nucleotide pairs . The term "hybridization" refers to the formation of a double structure by two double-stranded nucleic acids due to the pairing or formation of complementary base pairs. Hybridization can occur between fully complementary nucleic acid strands or between "substantially complementary" nucleic acid strands containing minor regions of mismatch. The conditions under which only fully complementary nucleic acid strands will hybridize will be referred to as "stringent hybridization conditions" or "specific hybridization conditions for a sequence". Stable duplicates of substantially complementary sequences can be achieved under less stringent hybridization conditions; the degree of tolerated mismatch can be controlled by the proper adjustment of the hybridization conditions. Those skilled in the art of nucleic acid technology can determine the stability of the duplicate by empirically considering several variables including, for example, the length and concentration of the base pairs of the nucleic acids. oligonucleotides, ionic strength, and the incidence of mismatched base pairs, following the guidance provided by the art, see, for example, Sambrook et al., 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor, New York; and Wetmur, 1991, Critical Review in Biochem. and Mol. Biol. 26 (3/4): 227-259). The term "primer" refers to an oligonucleotide capable of acting as a starting point of a DNA synthesis under the conditions in which the synthesis of a primer extension product with a strand of the nucleic acid is induced, i.e. , either in the presence of four different nucieoside triphosphates and an agent for extension (eg, a polymerase or DNA reverse transcriptase) in an appropriate buffer solution and at a suitable temperature. When used herein, the term "primer" is intended to encompass the oligonucleotides used in the binding-mediated amplification processes, in which one of the oligonucleotides is "extended" by binding to a second oligonucleotide which is hybridized to an adjacent position. Accordingly, the term "primer extension", when used herein, refers to both the polymerization of the individual nucleoside triphosphates using the primer as a Starting point of the DNA synthesis as to the binding of the primers to form an extended product. A primer is preferably a single-stranded DNA. The appropriate length of a primer depends of the proposed use of the primer but typically ranges from 6 to 50 nucleotides. Short primer molecules generally require colder temperatures to form sufficiently stable hybrid complexes r40m with the model or matrix. A primer does not need to reflect the exact sequence of the model nucleic acid, but it must be sufficiently complementary to hybridize with the model. The design of suitable primers for the amplification of a given target sequence or target is well known in the art and is described in FIG. literature cited here. The primers may incorporate additional features which allow the detection or immobilization of the primer but which do not alter the basic property of the primer, that of acting as a primer. point of beginning of the synthesis of the DNA. For example, the primers may contain an additional nucleic acid sequence at the 5 'end which does not hybridize to the target or target nucleic acid, but which facilitates the cloning of the amplified product. The primer region which is sufficiently complementary with the model for hybridization is referred to herein as the hybridization region. The terms "target or target", "target or target sequence", "target or target region", and "Target or target nucleic acid" refers to a region or subsequence of a nucleic acid to be amplified. When used here, a primer is' ^ t "specific" for a target sequence or target if the number of mismatches present between the primer sequence and the target or target sequence is less than the number of mismatches present between the primer sequence and the non-target or target sequences, which may be present in the sample. Hybridization conditions can be chosen so that stable duplicates are formed only if the number of mismatches present is not greater than the number of mismatches present between the primer sequence and the target sequence. objective. Under such conditions, the primer can form a stable duplicate with only a target or target sequence. Therefore, the use of specific primers for the target or target under properly stringent amplification conditions, makes it possible to specific amplification of those target or target sequences which contain the binding sites of the target or target primer. The use of specific amplification conditions for the sequence makes possible the specific amplification of those target or target sequences which contain the exactly complementary primer binding sites. The term "non-specific amplification" refers to the amplification of nucleic acid sequences other than target or target sequence which results from primers that hybridize to sequences other than the target or target sequence and which then serve as a substrate for the extension of the primer. Hybridization of a primer to a non-target or target sequence is referred to as "non-specific hybridization" and may occur during pre-amplification conditions, of reduced strict levels, of lower temperature. The term "primer-dimer" refers to a non-specific amplification product, independent of the model, which results from the primer extensions wherein another primer serves as a model. Although the primer-dimer often appears to be a binding agent of two primers, ie, a dimer, chaining agents of more than two primers may also be present. The term "primer-dimer" is used generically here to encompass the non-specific amplification product, independent of the model. The term "reaction mixture" refers to reagents that contain a solution, necessary for carry out a given reaction. An "amplification reaction mixture", which refers to a solution containing the reagents necessary to carry out an amplification reaction, typically contains primers Wn > of oligonucleotides and a polymerase or DNA ligase in a suitable buffer solution. A "PCR reaction mixture" typically contains oligonucleotide primers, a thermostable DNA polymerase, dNTPs, and a divalent metal cation in a suitable buffer solution. A mixture of the reaction is referred to as complete if it contains all the necessary reagents to make the reaction possible, and incomplete if it contains only a subset of the necessary reagents. It will be understood by a person with experience in the technique that the components of the reaction are routinely stored as separate solutions, each containing a subset of total components, for reasons of convenience, storage stability, or to allow application-dependent adjustment of concentrations of the component, and, that the components of the reaction are combined prior to the reaction to create a mixture of complete reaction. In addition, it will be understood by a person skilled in the art that the components of the reaction are packaged separately for commercialization and that useful sets or commercial sets can contain any subset of the reaction components which include the modified primers of the reaction. the invention.

Modified primers The amplification primers of the invention are modified by the covalent attachment of a group to one of the four nucleotides at the 3'-terminal end of the primer. In one embodiment, a modified primer of the invention consists of a sequence of nucleic acids having the general structure: R R I I 5'-S1-N-3 'or 5'-S1-N-S2-3', wherein Si represents a first nucleotide sequence between about 5 and about 50 nucleotides in length; wherein S2 represents a second sequence between one and three nucleotides in length; wherein N represents a nucleotide containing a purine or pyrimidine base containing an exocyclic amine; wherein R represents a modifying group, wherein R is covalently bonded to N via the exocyclic amine, and wherein R has the structure described below. As shown in the examples, the effect of the modification is maximized when the modification is with respect to the 3 'terminal nucleotide. Accordingly, preferably, the primer contains a modified 3 'terminal nucleotide. The modified nucleotide is selected from those whose base contains an exocyclic amine that is involved in mating or base pair formation with its complementary nucleotide. Typically, the primers are the DNA that contains only conventional nucleotides. Of the four conventional nucleotide bases found in DNA, adenine, guanine, and cytosine contain an exocyclic primary amine which is involved in the formation of base pairs with the complementary base. In the preferred aspect of the invention, the primer is modified by fixing a single modifying group to the exocyclic amine, substituting one of the two hydrogens of the amino group which, in the unmodified base, are able to be involved in the formation of base pairs. The structures of the modified nucleotides containing a base of adenine, guanine and modified cytosine, respectively, are shown below. wherein S represents the sugar portion, and R represents the modifying group. The present invention is not limited to primers consisting of conventional nucleotides. Any nucleotide analogue in which the base portion contains an exocyclic primary amine which is involved in the formation of base pairs with a complementary base is modifiable as described herein. Examples of non-conventional nucleotides include 3-methyladenine, 7-methylguanine, 3-methylguanine, 5-methyl cytosine, and 5-hydroxymethyl cytosine. The modifying group limits the ability of the modified base to participate in hydrogen bonding because the modifier subtitles a hydrogen atom. The remaining hydrogen atom can still participate in the hydrogen bonding. The modifiers can therefore influence both the kinetic characteristics and the thermodynamic characteristics of the hybridization. A variety of modifying groups are contemplated, which possess the following properties: 1. they interfere with, but do not prevent, the Watson-Crick base pair formation of the base modified with the complementary base; 2. interfere with, but do not prevent, the extension of the modified primer; and 3. allow the synthesis of a complementary strand with respect to the extension product of the modified primer.

The modifying group interferes sterically with the formation of the base pairs and, consequently, with the extension of the primer. Accordingly, the physical volume of the modifier influences the degree of interference with hybridization. When a modified adenosine or cytidine nucleotide protrudes into the central space of the major groove. Consequently, the modifying groups still relatively large must cause the small steric disturbance of the double structure. However, suitable modifiers are no longer large, such that the binding of the hydrogen is prevented or the enzymatic extension of the 3'-hydroxyl is prevented. When the modified guanosine nucleotide is incorporated into a double-stranded nucleic acid, the modifier group protrudes into the minor groove or slot, which provides less space to accommodate the volume of the modifier group. Accordingly, smaller modifying groups are preferred for attachment to a guanine base. The primer extension products, which are used as models in the subsequent amplification cycles, contain the modified base introduced by the primer. The modifier group is chosen such that the presence of the modified base in the model does not cause termination of primer extension or inhibition of primer extension. Preferably, the nature of the modifier group does not cause utaghenic events whereby the identity of the modified base is lost during the replication of a model derived from the primer. The effect of modified base in the model on primer extension can be routinely tested following the guidance provided herein and in the art (see, for example, Gniazdowski and Cera, 1996, Chem. Rev. 96: 619-634).

The modifier groups, R, which satisfy the above properties, are suitable for use in the methods of the present invention. Preferred modifier groups have the structure: R, -C-R2 H 2 wherein Ri and R2 independently represent hydrogen, an alkyl group with Ci-Cio, an alkoxy group, a phenyl group, a phenoxy group, a substituted phenyl group, a naphthyl group, or a substituted naphthyl group. The alkyl groups can be branched or linear. The largest alkyl groups, up to at least C20, can also be used. In the preferred embodiment, R cs a 2-naphthylmethyl group; a benzyl group; or a substituted benzyl group. Preferred substituted benzyl groups have the structure: wherein R3 represents a linear or branched alkyl group with C? -C6, more preferably a linear or branched alkyl group with C? ~ C, a methoxy group, or a nitro group. A linear or branched alkyl group with C? -C is methyl, ethyl, propyl, butyl, iso-propyl, isobutyl, tert-butyl and the like. Methyl and tert-butyl are the preferred alkyl groups. Preferably, R3 is fixed in the para position. The particularly preferred R modifier groups are shown below: benzyl p-methylbenzyl p-tert-butylbenzyl p-methoxybenzyl o-nitrobenzyl 2-naphthylmethyl Several of the particular modifier groups are described in the Examples. In general, an empirical selection of a suitable suitable modifying group from the class of the disclosed compounds can be routinely carried out by one skilled in the art following the guidance provided herein. Preferably, the suitability of a particular group is determined empirically by the use of the modified primers in the amplification reaction. Successful amplification indicates both that the modified base does not fully inhibit the extension of the primer, and that the presence of the modified base in a model derived from the primer does not cause termination of the primer extension. The reduction of the primer-dimer is determined as described in the Examples.

Primers with a Photo-Labile Modification In an alternative embodiment of the invention, the primers are modified with one or more photolabile groups which can be removed by exposure to light after the reaction has reached the reaction conditions at elevated temperature which assure specificity. Because the modifier is removed prior to the extension of the primer, the modified primer need not be extendable prior to the removal of the group. Examples of the photolabile modifiers that can be used in the methods of the present invention are described in Pillai, 1980, "Photoremovable Protecting Groups in Organic Synthesis", Synthesis: 1-26.

Preferably, the photolabile primers of the invention are modified at the 3 'terminal nucleotide by the attachment of one or two groups of o-nitrobenzyl: In the primers modified by the attachment of a single nitrobenzyl group to the primary exocyclic amine of a base portion, the resulting secondary amine can still participate in base pair formation if the amine group is rotated in such a way that hydrogen remaining is oriented towards the complementary base. As described in the examples, these primers can be used in an amplification with or without removal by irradiation with UV light. Primers modified by the attachment or joining of groups of two nitrobenzyls to the exocyclic amine of the base can not be extended. The inhibition presumably results from the inability of the modified base to undergo the formation of the base pairs, which is avoided because both hydrogens of the exocyclic amine are replaced by the bulky nitrobenzyl groups. The use of the modified primers with two nitrobenzyl groups in one Amplification, in which the reaction mixture was exposed to UV light for a period of time sufficient to remove the nitrobenzyl groups, whereby primer extension is allowed to be carried out, is described in the Examples. In an alternative embodiment, the modifier group is fixed to the ring nitrogen. The primers modified by the attachment of a nitrobenzyl group to the nitrogen of the base ring can not be extended due to the inability of the modified base to undergo the formation of base pairs. Removal of the nitrobenzyl groups by exposure to ultraviolet light allows primer extension to take place. The use of the photo-labile primers which can not be extended until the modifier group is essentially removed, provides a "hot start" amplification. The extension of the primer is inhibited during non-specific pre-reaction conditions. The reaction is irradiated and the primers unblocked only after the temperature of the reaction has been raised to a temperature which ensures the specificity of the reaction.

• Synthesis of the Modified Primers The synthesis of the modified primers is carried out using standard chemical means well known in the art. The methods for the introduction of these modifiers can be divided into four classes. "1. The modifier can be introduced through the use of a modified nucleoside as a support for the synthesis of DNA. 2. The modifier can be introduced by the use of a modified nucleoside such as a phosphoramidite. 3. The modifier can be introduced by means of the use of a reagent during DNA synthesis. (For example, benzylamine treatment of a convertible amidite when incorporated into a DNA sequence). 4. Post-synthetic modification. The modifier can be introduced as a reagent when contacted with the synthetic DNA.

The synthesis of the particular modified primers is described in the Examples. The primers additional modifications can be synthesized using standard synthesis methods in an analogous way. Preferably, the modified primers are synthesized using a support for glass synthesis of controlled pore size, derivative (CPG). A general reaction scheme for the synthesis of the CPG of the derivatized dA is shown in Figure 6. Particular modifying groups can be added through the use of the alkyl halide, benzyl halide, substituted benzyl halide, methylnaphthyl halide, or substituted methylnaphthyl-halide alkylating agent, suitable. The synthesis of the CPG of benzyl- and p-tert-butylbenzyl-dA described in Examples 1 and 2 follows the scheme shown in Figure 6. The alkylation of the exocyclic amino group can be carried out using methods analogous to the methylation described in Griffin and Reese, 1963, Biochim. Act 6_8: 185-192. Additional synthesis methods are described in Aritoma et al., 1995, J. Chem. Soc. Perkin Trans. 1: 1837-1849.

Amplifications using the Modified Primers The methods of the present invention comprise carrying out an amplification based on a primer, using the modified primers of the present invention. invention. In general, the modified primers can be replaced by unmodified primers containing the same nucleotide sequence in a primer-based amplification, without change in the conditions of the amplification reaction. Of course, a person skilled in the art will recognize that minor reoptiinization of routine reaction conditions may be beneficial in some reactions. In a preferred embodiment, the modified primers of the present invention are used in the polymerase chain reaction (PCR). However, the invention is not restricted to any particular amplification system. The modified primers of the present invention can be used in any amplification system based on a primer in which the primer-dimer or the non-specific amplification product can be formed. Examples include the amplification methods described in the references cited above. When other systems are developed, these systems can benefit by the practice of this invention. The methods of the present invention are suitable for the amplification of either DNA or RNA. For example, RNA amplification using a polymerase chain reaction / reverse transcription (PCR-RT) is well known in the art and is described in U.S. Pat. Nos. 5,322,770 and 5,310,652, Myers and Gelfand, 1991, Biochemistry 3_0 (31): 7661-7666, Young et al., 1993, J. Clin. Microbiol. 31 (4): 882-886, and Mulder et al., 1994, J. Clin. Microbiol. 32 (2): 292-300. In a primer-based amplification, the extension of the primer is typically carried out at an elevated temperature using a thermosetting enzyme such as a thermostable DNA polymerase. The enzyme initiates the synthesis at the 3 'end of the primer and proceeds in the direction towards the 5' end of the model until the synthesis is completed. Purified, thermostable DNA polymerases useful in amplification reactions are well known in the art and include, but are not limited to, the enzymes described in U.S. Pat. No. 4,889,818; U.S. Pat. No. 5,079,352; U.S. Pat. No. 5,352,600; U.S. Pat. No. 5,491,086; WO 91/09950; WO 92/03556; WO 92/06200; WO 92/06202; WO 92/09689; and U.S. Pat. No. 5,210,036. A review of the thermostable DNA polymerases is provided in Abramson, 1995, in PCR Strategies, (ed. M.A. Innis et al.), Academic Press, San Diego. In a preferred embodiment, particularly for the amplification of DNA, the amplification is carried out using a reversibly inactivated enzyme as described in the use of a reversibly inactivated enzyme, which is reactivated under the conditions of the reaction at elevated temperature, in addition the non-specific amplification is reduced by inhibiting the extension of the primer prior to the start of the reaction. A reversibly inactivated thermostable DNA polymerase, developed and manufactured by Hoffmann-La Roche (Nutley, NJ) and marketed by Perkin Elmer (Norwalk, CT), is described in Birch et al., 1996, Nature 381 (6581): 445- 446 The effect of the modifying group on the ability of the enzyme to extend the primer depends, in part, on the particular enzyme used and, in part, on the selected reaction conditions. For example, the DNA polymerase T is most permissive when the Mn + 2 is used as the divalent cation, as in some RNA amplifications, instead of Mg2 + '. A person skilled in the art will recognize that in the routine selection of a suitable modifying group, the enzyme and reaction conditions will be considered. Sample preparation methods suitable for the sections of the amplification are well known in the art and are fully described in the literature cited herein. The particular method used is not a critical part of the present invention. A A person skilled in the art can optimize the reaction conditions for use with the known sample preparation methods. Methods of analyzing the amplified nucleic acid are well known in the art and are fully described in the literature cited herein. The particular method used is not a critical part of the present invention. A person skilled in the art can select an appropriate analysis method depending on the application. A preferred method for analyzing an amplification reaction is by verifying the increase in the total amount of double-stranded DNA in the reaction mixture, as described in Higuchi et al., 1992, Bio / Technology 10: 413-417; Higuchi et al., 1993, Bio / Technology 1: 1: 1026-1030; European Patent Publications Nos. 512,334 and 640,828. In this method, referred to herein as "kinetic PCR", the detection of double-stranded DNA is based on the increased fluorescence exhibited by etididium bromide (EtBr) and other DNA binding tags when they bind to double-stranded DNA. The amplification is carried out in the presence of the label. The increase in double-stranded DNA resulting from the synthesis of the target or target sequence leads to a detectable increase in fluorescence, which is verified during the amplification. Therefore, the methods make it possible to verify the progress of the amplification reaction. In a kinetic PCR, the measured fluorescence depends on the total amount of double-stranded DNA present, whether resulting from non-specific amplification or amplification of target or target sequence. Fluorescence verification allows measurement of the increase in the total amount of double-stranded DNA, but the increase that results from the amplification of target or target sequence is not measured independently of the increase that results from the non-specific amplification product. The modified primers of the present invention are particularly useful in kinetic PCR because they not only reduce the amount of primer-dimer formed, but also retard the formation of detectable amounts of the primer-dimer. A delay in the formation of the primer-dimer until after a significant increase in target or target sequence has occurred, makes it possible to independently verify the amplification of target or target sequences and minimizes primer-dimer interference.

Games or Sets The present invention also relates to sets or sets, multiple container units comprising components useful for the practice of the present method. A useful set or kit contains the primers, at least one of which is modified as described herein, for the amplification of the nucleic acid. Other optional components of the kit or set include, for example, an agent to catalyze the synthesis of the primer extension products, the nucleoside triphosphates of the substrate, the appropriate buffer solutions of the reaction, and instructions for carrying out the present method. The examples of the present invention presented below are provided for illustrative purposes only and do not limit the scope of the invention. Numerous embodiments of the invention within the scope of the claims following the examples will be apparent to those of ordinary skill in the art from the reading of the preceding text and the following Examples.

Example 1 Synthesis of Modified Primers with a Benzyl Group The primers modified by the addition of the benzyl group were synthesized by one of two processes, described below. The primers modified at the 3 'terminal base were synthesized using the Controlled Pore Size Glass (CPG) of Nd-benzyldeoxydadenosine to initiate DNA synthesis. The primers modified in an internal base were synthesized using a phosphoramidite of N5-benzyldeoxydadenosine. The following standard abbreviations are used in the example: DMAP 4-Dimethylaminopyridine DMF N, N-Dimethylformamide TEA Triethylamine EDC L-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride THF Tetrahydrofuran DMT 4,4'-Dimethoxytrityl LCAA-CPG Controlled pore size glass of Alkyl Amino Long chain Synthesis of N6-benzyldeoxydadenin CPG Step 1: Synthesis of N -benzoyl, N -benzyl, 5'-0-DMT-2 '- deoxyadenosine To the N6-Benzoyl-5'-0- (4,4'-dimethoxytrityl) -2'-deoxyadenosine (657 mg, 1.0 mmol, Aldrich Chemical Co., Milwaukee, Wl), pyridine (10 mL) is added and the The mixture is dried by evaporation under vacuum. This is repeated. The resulting foam is dissolved in anhydrous DMF (15 ml, Aldrich Chemical Co., Milwaukcc, Wl) and cooled to 5 ° C. Sodium hydride (44 mg, 1.1 moles, 1.1 equivalents 60% dispersion in oil) is added under an argon atmosphere and stirred at room temperature for 45 minutes. The benzyl bromide (143 μl, 206 mg, 1.2 mmol, 1.2 equiv, Aldrich Chemical Co., Milwaukee, Wl) was added for 2 minutes and the mixture was stirred overnight at room temperature. The mixture is dried by evaporation under vacuum and the residue is partitioned between ethyl acetate and water (10 ml each) and extracted. The aqueous phase is back extracted with ethyl acetate (10 ml) and the combined extracts are dried over anhydrous magnesium sulfate, filtered and evaporated. The crude product is purified by column chromatography on silica gel (75 g) using methanol, triethylamine, methylene chloride (3: 0.5: 96.5). The fractions that containing the product were combined and dried by evaporation to give the N6-benzoyl, N6-benzyl, 5'-O-DMT-2 '-deoxyadenosine expected (410 mg, 54%). The structure of the product was confirmed by NMR.

Step 2: Succiniiación To the N6-benzoyl, N6-benzyl, 5'-0-DMT-2'-deoxyadenosine (295 mg, 0.39 mmol), pyridine (10 ml) is added and the mixture is dried by evaporation under high vacuum. This step was repeated. The fresh anhydrous pyridine (10 ml) is added together with the succinic anhydride (200 mg, 2 mmol, 5.0 equiv.) And DMAP (24 mg), and the solution is stirred under an argon atmosphere overnight at room temperature. The volume of the solvent was stirred under vacuum and the residue sc partitioned between methylene chloride (20 ml) and sodium citrate solution (20 ml, 0.1 M, pH 5.0) and extracted. The aqueous phase was extracted with more methylene chloride (20 ml) and the combined extracts were dried over anhydrous sodium sulfate, filtered, and dried by evaporation. The product is purified by column chromatography on silica gel (4.5 g) using ethyl acetate, triethylamine, methylene chloride (32: 1: 67) to give the expected 3'-succinate ester of N6-benzoyl- N6- benzyl-3'-O-succinate-5 '-O-DMT-2' -deoxy-adenosine (247 mg, 74%).

Step 3: Referral of the CPG The CPG of the acid wash is prepared as follows. LC7? A-CPG (1.0 g, LCA00500C, 500-angstroms, 88.6 (moles / g; CPG Inc., Fairfield, NJ) was washed with dichloroacetic acid in dichloromethane (2%, 20 ml) by swirling it periodically for 20 minutes at room temperature The acid washed CPG was filtered on a glass frit and washed with dichloromethane until it was acid free, the powder was air dried, then dried under vacuum at room temperature overnight. The binding of the modified nucleoside intermediate ai CPG washed with acid was carried out as follows: To a solution of the N6-benzoyl-N6-benzyl-3 '-0-succinate-5' -O-DMT-2 '-deoxy-adenosine (170 mg, 0.2 mmol), prepared as described above, in dichloromethane (10 ml) is added TEA (100 μl), and the solution is concentrated to about 5 ml under an argon atmosphere DMAP (12 mg, 0.1 mmoles, 0.5 equiv.), TEA (100 μl), EDC (384 mg, 2.0 mmol, 10 equiv.), and the CPG washed with acid from above, they added cn sequence. Sc added anhydrous pyridine (5 ml) and the mixture is sealed and stirred for 3 days at room temperature. The CPG is removed by vacuum filtration and washed extensively with isopropanol, then with dichloromethane, air dried, then dried under vacuum for 1 hour. The coronation at the ends of the derived CPG is carried out as follows. To the dry derivatized CPG, the solutions of Cap A and Cap B (5 ml of each, Acetic Anhydride / 2, 6-Lutidine / THF and 10% of N-Methylimidazole in THF) are added.; DNA synthesis reagents from Glen Research, Sterling, VA) and the mixture is stirred for 4 hours at room temperature. The CPG is removed by vacuum filtration and washed extensively with isopropanol, then with dichloromethane, air-dried sc, then dried under vacuum overnight.

II. Synthesis of N6-Benzyl Phosphoramidite Deoxyadenosine N6-benzoyl, N6-benzyl, 5'-0-DMT-2'-deoxyadenosine was synthesized as described above. To N6-benzoyl, N6-benzyl, 5'-O-DMT-2'-deoxyadenosine (196 mg, 0.26 mmol) in dry THF (8 ml) is added diisopropylethylamine (350 μl, 270 mg, 2.04 mmol, 7.8 equiv. .) and the N, N- 2-cyanoethyl diisopropylchlorophosphoramidite (161 mg, 0.68 mmol, 2.6 equivalents, Aldrich Chemical Co., Milwaukee, Wl), and the mixture is stirred for 30 minutes at room temperature under an argon atmosphere. The solvent was removed under vacuum and the residue was partitioned between a solution of sodium bicarbonate (5%, 20 ml) and ethyl acetate (20 ml). The organic phase is washed with the bicarbonate solution, water, and saturated brine (20 ml each in sequence, dried over sodium sulfate, filtered, and evaporated) The residue is purified by column chromatography on silica gel. (4 g) using acetone / hexane / TEA (34: 65: 0.7) to give the desired phosphoramidite (248 mg, 100%).

III. Synthesis, purification and DNA analysis.

The CPG of adenosine derived from benzyl (25 mg, 1.0 (mol) was transferred to the empty synthesis columns (Fien Research, Sterling, VA) and these were used to make oligonucleotides on an ABI 374 DNA synthesizer (Perkin Elmer, Applied Biosystems Division, Foster City, CA) using the conventional conditions of synthesis and deprotection.The DMT-? DN crude sc was purified and sc converted to 5 '-hydroxy-DNA by the HPLC On / Off of standard DMT using a DNA column -Puro Rainin on a CLAR system from Rainin (Rainin Instrument Co, Woburn, MA). The oligonucleotides were analyzed using an ABI capillary electrophoresis system (Perkin Elmer, Applied Biosystems Division, Foster City, CA) or by denaturing anion exchange CLAR chromatography on a Dionex Nucleopak column (Dionex Corp, Sunnyvale, CA). Similarly, the synthesis of the internally modified primers was carried out using an unmodified CPG and the modified phosphoramidite synthesized as above.

Example 2 Synthesis of primers modified with a t-butyl-benzyl group The present example describes the synthesis of the modified primers in terminal 3 'adenosine with a p-tert-butylbenzyl group. The modified primers were synthesized essentially as described in Example 1, but using the CPG of NG- (p-tert-buti-benzyl) deoxyadenosine. The synthesis of the derived CPG is described later.

Step 1: Synthesis of N -benzoyl-N - (p-tert-butylbenzyl) -5 '-0- (4,4'-dimethoxytrityl) -2'-deoxyadenosine To the N6-benzoyl.l-5 '-0- (4,4'-dimethoxytrityl) -2'-deoxyadenosine (658 mg, 1.0 mmol) is added DMF (anhydrous, 10 ml) and evaporated to dryness. This was repeated. The fresh DMF (10 ml) is added under an Argon atmosphere. Sodium hydride (44 mg, 60% in oil, 1.1 mmol) is added and the mixture is stirred for 0.5 hour at room temperature. The 4- (tert-butyl) benzyl bromide (272 mg, 1.2 mmol) is added dropwise and stirred at room temperature overnight. The solvent is removed under vacuum, and the residue is partitioned between ethyl acetate and water (20 ml each). The organic phase is washed with water (3 times, - 20 ml), dried over anhydrous magnesium sulfate, filtered and evaporated to dryness. The crude product is purified by column chromatography on silica gel (100 g), using methylene chloride: methanol: triethylamide 96.5: 3.0: 0.5 to give the N6-benzoyl-N6- (p-tert-butylbenzyl) - 5'-O- (4,4'-dimethoxytrityl) -2'-deoxyadenosine, (229 mg, 28.5%).

Step 2: Succinylation.

N 6 -benzoyl-N 6 - (p-tert-butylbenzyl) -5 '-0- (4,4'-dimethoxytrityl) -2'-deoxyadenosine (217 mg, 0.27 mmol) is treated with succinic anhydride (135 mg, 5%). equiv.) and DMAP (17 mg, 0.5 equiv.) in pyridine (10 ml). The working procedure and the chromatography as described in Example 1, above, gave the 3'-0-succinate of N6-benzoyl-N6- (p-tert-butylbenzyl) -5'-O- (4, 4 ' dimethoxytrityl) -2'-deoxyadenosine (199 mg, 82%).

Step 3: Referral of the CPG The succinate (180 mg, 0.2 mmol) of step 2, above, was treated with the LCAA-CPG washed with an acid (as described in Example 1). The CPG was capped at the ends and dried in vacuo to give the CPG derived from the 3'-N-benzoyl-N6- (p-tert-butylbenzyl) -5 '-O- (4, 4' - dimethoxytritii) -2'-deoxyadenosine, (1065 g).

Example 3 Synthesis of Modified Primers with a methyl group The primers modified in the 3'-terminal adenosine with a methyl group were synthesized using the CPG of N6-methyl dA (22 mg, 1 μmol, Glen Research, Sterling VA). The N6-methyl dA CPG was placed on an empty synthesis column, and the primers were made according to the standard conditions of synthesis and deprotection. The primers were purified using the CLT On / Off method of DMT as described in Example 1.

Example 4 Synthesis of Photo-Labile Modified Primers The present example describes the synthesis of modified primers in terminal 3 'adenosine with either one or two nitrobenzyl groups. The modified primers were synthesized essentially as described in Example 1, but using either a CPG of mononitrilebenzyl dA or a CPG of bis-nitrobenzyl dA.

I. Mononitrobenzylated primers The general method for the synthesis of the CPG derived from N6-benzoyl-N6-benzyl-2'-deoxyadenosine (see Example 1) was applied to the synthesis of the CPG derived from Nb-benzoyl-Nb-orthonitrobenzyl-2 '- deoxyadenosine, by the replacement of orthonitrobenzyl bromide as the alkylating agent. Subsequent steps for the CPG were identical to those described in Example 1, with the addition that the intermediates were protected from ambient light by wrapping the reaction flasks in an aluminum foil. Following synthesis of the derived CPG, the primers were synthesized as described in Example 1, but were isolated by solid phase extraction using the Nensorb Prep disposable columns (NEN Research Products Biotechnology Systems, Du Pont Co, Boston MA ), using the protocols that were described by the manufacturer.

II. Bis-nitrobenzylated primers The GPC of bis-nitrobenzyl deoxyadenosine was synthesized as described below. Following the synthesis of the derived CPG, the primers were synthesized and purified as described for the mononitrobenzyl primers.

Step 1: Synthesis of 5'-0-DMT-N6-bis-ort? -nitrobenzyl-2'-deoxyadenosine The 2'-deoxyadenosine monohydrate (538 mg, 2.0 mmol, Aldrich Chemical, Milwaukee, Wl) was dried by evaporation with anhydrous pyridine (2 times, 10 ml) under vacuum. The residue is dissolved in anhydrous DMF (10 ml, Aldrich, Milwaukee, Wl) under an argon atmosphere, and sodium hydride (88 mg, 2.2 mmol, 1.1 equivalents, 60% dispersion in oil) is added and stirred during 40 minutes at room temperature. Sc adds 2-nitrobenzyl bromide (710 mg, 3.3 mmol, 1.5 equivalents) and the solution is stirred for 4 hours at room temperature. The DMF is removed by evaporation under vacuum and the residue is partitioned between ethyl acetate and water (20 ml each). The aqueous phase is extracted with ethyl acetate (20 ml) and the combined extracts are washed with water (20 ml) and dried over magnesium sulfate, filtered and evaporated. The crude product is purified by column chromatography on silica gel (50 g, using 3% MeOH in CH 2 Cl 2) to give 2'-deoxy-N 6 -bis-ortho-nitrobenzyladenosine (320 mg, 30%). To the 2'-deoxy-Nb-bis-ortho-nitrobenzyladenosine (200 mg, 0.518 mmol) anhydrous pyridine (10 ml) is added and evaporated to dryness. Pyridine (10 ml) is added followed by 4-4'-dimethoxytrityl chloride (900 mg, 2. 3 mmol, 4.5 equiv.) And triethylamine (280 mg, 2.76 mmol, 4.0 equiv.) And stirred at room temperature under an argon atmosphere for 5 hours. Water (0.5 ml) is added and stirred for 20 minutes. The mixture is partitioned between ether and water (20 ml each) and the aqueous phase is extracted again with ether (20 ml). The extracts were combined and washed with water (20 ml) and sc dried over anhydrous sodium sulfate., sc filter and evaporate. The material was purified by chromatography on silica gel (4 g, using 0.7-2.5% methanol in methylene chloride) to give the 5'-0-DMT-N6-bis-ortho-nitrobenzyl-2'-deoxyadenosine (121 mg, 33%).

Step 2: Succinylation The 5'-0-DMT-N6-bis-ortho-nitrobenzyl-2'-deoxyadenosine (121 mg, 0.145 mmol) was dried by evaporation with anhydrous pyridine (2 times, 3 ml). Pyridine (3 ml), succinic anhydride (58 mg, 0.58 mmol, 4 equiv.) And DMAP (11 mg, catalytic) were added, and the solution was stirred at room temperature for 3 days. The solution was evaporated in vacuo, and the residue was partitioned between methylene chloride (10 ml) and the sodium citrate buffer (0.1 M, pH 5.0, 10 ml). The organic phase is dried over anhydrous sodium sulfate, filtered and evaporated to dryness. The raw product is purified by chromatography on silica gel (2 g, using EtOAc: CH2C12: TEA, 32: 67: 1 10 ml, then MeOH: CH2Cl2, 3:97, 25 ml) to give a faint yellow foam, 3 ' -O-succinate of 5'-0-DMT-Nb-bis-ortho-nitrobenzyl-2'-deoxyadenosine (138 mg, 99.5%).

Step 3: Referral of the CPG LCAA-CPG washed with acid was prepared as in Example 1. The binding of the modified nucleoside intermediate to the washed CPG with acid was carried out as follows. the 3 '-O-succinate of 5'-0-DMT-N6-bis-ortho-nitrobenzyl-2'-deoxyadenosine (37 mg, 0.04 mmol) was treated with TEA (16 (1) in a colored glass vial amber, and evaporated.To this residue is added anhydrous pyridine (1.5 ml), TEA (2 μl), DMAP (2.4 mg), EDC (76 mg, 0.04 mmol) and LCAA-CPG washed with acid (200 mg). and the mixture is stirred in an orbital mixer for three days at room temperature.The CPG was removed by filtration under reduced pressure and washed extensively with isopropanol, then with methylene chloride, dried with air, then dried under vacuum during 1 hour The coronation at the ends of the derived CPG was carried out as described in Example 1.

Example 5 Amplifications using Modified Primers - Effect of Modified Nucleotide Position To demonstrate the effect of the modified primers on primer-dimer formation, comparisons of the HIV-1 RNA amplifications were carried out using both modified primers and unmodified primers. In addition, to evaluate the effect of the position of the modified nucleotide on the reduction of the primer-dimer, amplifications were carried out using three different upstream modified primers, which differ only in the location of the modified base.

Nucleic Acid of Target or Target The HIV-1 RNA models were synthesized using an HIV-1 RNA transcription vector essentially as described in Mulder et al., 1994, J. Clin. Microbiol. 32 (2): 292-300.

Primers The amplifications were carried out using both modified and unmodified primers. The nucleotide sequences of the unmodified primers are shown below, oriented in the 5 'to 3' direction. The current primer RAR1032MB (SEQ ID NO: 1) and the downstream primer RAR1033MB (SEQ ID NO: 2) amplify a product of 175 base pairs corresponding to positions 2956 to 3130 of the nucleotide, of the sequence of the reference strain HIV-1 HXB2 (GenBank Accession No. K03455).

HIV-l Amplification Primers Primer Seq. ID No. Sequence RAR1032MB 1 CAATGAGACACCAGGAATTAGATATCAGTACAA RAR1033MB 2 CCCTAAATCAGATCCTACATATAAGTCATCCA The above primer designations refer to the unmodified primers. Unmodified primers were biotinylated at the 5 'end. The modified primers were synthesized as described in Example 1, which consisted of the same nucleotide sequences as the unmodified primers, but containing a benzylated adenosine either at the 3 'terminal position or at a position one to three nucleotides upstream of the 3' end. The modified forms of the primers are designated here as follows: Amplification Primers Modified with V? H-1 Primer Seq. Id. No. Position of Modified Nucleotide RAR1032MBA1 1 3 'terminal RAR1032MBA2 1 1 from 3' terminal RAR1032MBA4 1 3 from 3 'terminal RAR1033MBA1 2 3' terminal Amplification Amplifications were carried out in 100 μl reactions containing the following reagents: 100 copies of RNA from the 50 mM HIV model of Tricine (pH 8.33), 110 nM KOAc, 300 μM each of dATP, dCTP, and dGTP , 50 μM dTTP 500 μM dUTP, 50 μM of each primer, 3.5 mM of Mn (OAc) 2, 13% Glycerol 20 units of DNA polymerase Z05 *, and 2.0 units of UNG **. * described in U.S. Pat. No. 5,455,170 ** manufactured and developed by Hoffmann-La Roche and marketed by Perkin Elmer, Norwalk, CT.

The cyclization of the amplification temperature was carried out in a thermal cyclisation apparatus of the TC480 DNA (Perkin Elmer, Norwalk, CT) using the following temperature profile: Incubation of the Pre-reaction 45 ° C for 4 minutes; Transcription-Reverse 60 ° C for 20 minutes; 46 cycles: denaturation at 94 ° C for 45 seconds, extension / annealing at 60 ° C for 45 seconds; Final extension 60 ° C for 7 minutes; Maintenance of 10 ° C until post-reaction analysis (for a short time).

Amplified Product Detection The presence of the amplified product was detected by gel electrophoresis as follows. The products of the reaction were fractionated using an agarose gel (100 ml of NuSieve at 3% and 0.5% SeaChem) and a current buffer solution of IX TBE (0.089 M Tris, 0.089 M boric acid, 0.0025 M EDTA disodium) was used. Ethidium bromide (0.5 μg / ml) was added to stain any DN present. The sc electrophoresis was carried out at 100 volts for about 1 hour. The ethidium bromide stained bands of the DNA were visualized using UV irradiation.

Results The results of the electrophoretic analysis of the gel are shown in Figure 1. The numbers of the strips or intervals corresponding to each of the amplifications using the combinations of the modified and unmodified primers are shown in the table given below. . The bands corresponding to the proposed HIV product are indicated in the figure by an arrow. The other bands in the gel correspond to a non-specific amplification product and, in particular, the primer-dimer.

Faj primers to No. Current Upstream Abaj or RAR1032MB RAR1033MB 1 RAR1032MBA1 RAR1033MB 2 RAR1032MB A2 RAR1032MBA4 RAR1032MBA4 RAR1033MB 4 RAR1032MB RAR1033MBA1 5 RAR1032MBA1 RAR1033MBA1 6 RAR1032MB A2 RAR1033MBA1 7 RAR1032MBA4 RAR1033MBA1 8 Because the formation of the primer-dimer is a matter of the formation of the proposed amplification product, a reduction in the primer-dimer typically leads to a concomitant increase in the amount of the proposed product formed. Accordingly, the effect of the modified primers can be observed by comparing the amount of the primer-dimer formed relative to the amount formed using the unmodified primers and by comparing the amount of the target or proposed target formed relative to the amount formed using the primers. not modified. A comparison of the results using two unmodified primers (strip 1) with the results using a unique 3'-modified primer (strips or intervals 2 and 5) and with the results using two 3'-modified primers (strip 6) indicate that a Decrease in the primer-dimer was obtained using either one or two modified primers. In the amplifications using a unique 3'-modified primer, a small difference in primer-dimer reduction was observed, which depends on which primer was modified. The use of two modified primers (band 6) led to both a greater reduction in the primer-dimer and a detectable increase in the amount of target sequence or amplified target. The effect of the position of the modified nucleotide is observed in a comparison of the bands or intervals 6-8. The reduction of the primer-dimer obtained using a modified primer in the nucleotide adjacent to the 3 'terminal nucleotide (band 7) was equivalent to that obtained using a modified primer in the 3' terminal nucleotide (band 6), whereas the improvement obtained using a primer modified at the bases of three nucleotides upstream of the 3 'terminal nucleotide (band 8) was slightly lower.

Example 6 Additional Amplification using Modified Primers Effect of Modified Nucleotide Position 5 To further demonstrate the effect of the modified primers on primer-dimer formation, comparisons of VCII RNA amplifications were made using both the modified primers and the unmodified primers. , essentially as described above. The amplifications were carried out using three different modified downstream primers, which differed only in the location of the modified base. White Nucleic Acid or Target "T VCH RNA models were synthesized using the VCH RNA transcription vector as described in Young et al., 1993, J. Clin. Microbiol. 31 (4): 882-886 .

Primers Amplifications were carried out using both modified and non-primed primers modified. The nucleotide sequences of the unmodified primers are shown below, oriented in the 5 'to 3' direction. The upstream primer ST280A (SEQ ID NO: 3) and the downstream primer S 778AA (SEQ ID NO: 4) amplify a product of 240 base pairs from the 5'-untranslated or translational region of the IICV genome.

HCV Amplification Primers Primer Seq Id. No. Nucleotide Sequence ST280A 3 GCAGAAAGCGTCTAGCCATGGCGTTA ST778AA 4 GCAAGCACCCTATCAGGCAGTACCACAA The designations of the above primer refer to the unmodified primers. The modified primers were synthesized as described in Example 1, which consisted of the same nucleotide sequences as the unmodified primers, but had a benzylated ade ssin either in the 3 'terminal position or in a one to three nuclotic position upstream of the 3 'end. The modified forms of the primers are designated here as follows: Modified VCH Amplification Primers Seq Primer Id. No. Modified Nucleotide Position S 280ABA1 3 3 'end ST778AABA1 4 3' end ST778AABA2 4 1 from the 3 'end ST778AABA4 4 3 from the 3' end Amplification and Analysis The amplifications were carried out essentially as described in Example 3, but using 100 copies of the CVH RNA model. The gel analysis of the amplified product was carried out as described in Example 3.

Results The results of the electrophoretic analysis of the gel are shown in Figure 2. The numbers of the strips or intervals corresponding to each of the amplifications using the combinations of the modified and unmodified primers are shown in the table below. The bands corresponding to the proposed VCII product are indicated in the figure by an arrow. The other bands in the gel correspond to the non-specific amplification product and, in particular, the primer-dimer.

Cebadore! S Faj a No. Current Upstream Abaj or ST280A ST778AA 1 ST280A ST778AABA1 2 ST280A ST778AABA2 3 ST280ABA ST778AABA4 4 ST280ABA1 ST778AA 5 ST280ABA1 ST778AABA1 6 ST280ABA1 ST778AABA2 7 ST280ABA1 ST778AABA4 8 Because the formation of the primer-dimer is a matter of the formation of the proposed amplification product, a reduction in the primer-dimer typically leads to a concomitant increase in the amount of the proposed product formed. Accordingly, the effect of the modified primers can be observed both by comparing the amount of the primer-dimer formed relative to the amount formed using the unmodified primers and by comparing the amount of the proposed target or target formed relative to the amount formed using the unmodified primers. The results obtained were similar to those obtained from HIV amplifications described in the previous example, but in the VCH amplifications, the increase in the proposed product was more evident than in the amplifications of HIV. A comparison of the results using two unmodified primers (strip 1) with the results using a unique 3'-endified primer (strips or intervals 2 and 5) and with the results using two 3'-modified primers (strip 6) indicate that a decrease in the primer-dimer was obtained using either one or two modified primers. The use of two modified primers (Strip 6) led to both the greatest reduction in the primer-dimer in the company of a significant increase in the amount of target sequence or amplified target. As in the previous example, a small difference in the reduction of the primer-dimer in the amplifications is observed using a unique 3'-modified primer that depends on which primer was modified. The effect of the position of the modified nucleotide is observed in a comparison of the strips or intervals 6-8. Essentially equivalent results are obtained using primers modified at the 3'-terminus nucleotide (girdle 6), the nucleotide adjacent to the 3'-terminus nucleotide (girdle 7), and the bases of three nucleotides upstream of the 3'-terminus nucleotide (girdle) 8). These results indicate that the group modifier can be fixed to any of the four nucleotides at the 3 'end of the primer.

Example 7 Amplifications using the Modified Primers - Effect of the Modifier Group To further demonstrate the effect of the modified primers on primer-dimer formation, and to demonstrate alternative modifications of the primer, comparisons of the VCH RNA amplifications were carried out using both the modified primers and the unmodified primers, where the primers were modified by the addition of one of three different modifier groups: benzyl, nitrobenzyl, and methyl groups. The results of the amplification were analyzed by two different methods. In a set of comparisons, the presence of the primer-dimer was evaluated by the eiectroforético analysis of the products of the reaction. In a second set of comparisons, primer-dimer formation was verified during amplification using the kinetic PCR methods described above.

Nucleic Acid of Target or Target HCV RNA models were synthesized using a VCH RNA transcription vector as described in Young et al., 1993, J. Clin. Microbiol. 31 (4): 882-886.

Amplification primers The amplifications were carried out using both modified and unmodified primers. The modified primers consisted of the same nucleotide sequences as the unmodified primers, but were modified into 3 '-terminal adenosine by the addition of a methyl group, a benzyl group, or a nitrobenzyl group. The primers were synthesized as described in the previous examples. The designation for the primers used is shown below.

Primer Seq Id.No. Modification of Base 3 'ST280A 3 unmodified ST280AMEA1 3 methyl ST280ABA1 3 benzyl ST280ANBA1 3 nitrobenzyl ST778AA 4 unmodified ST778AAMEA 4 methyl ST778AABA1 4 benzyl ST778AANBA1 4 nitrobenzyl Amplification Reactions The amplifications were carried out in 100 μl reactions containing the following # 10 reagents: 0, 20, or 200 copies of the 50 mM VCH RNA model of Tricine, pH 8.3 3. 5 mM Mn (OAc) 2; 15 300 μM each of dATP, dCTP, dGTP; 50 μM dTTP; 500 μM dUTP; 250 nM each primer; 20 U rTth *; 20 2U UNG *; and 13% Glycerol. * manufactured and developed by Hoffmann-La Roche and marketed by Perkin Elmer, Norwalk, CT. 25 The thermal cyclization of each reaction mixture was carried out in a GeneAmpRPCR System 9600 thermal cycler device (Perkin Elmer, Norwalk, CT) using the following temperature profile: incubation of the pre-reaction 45 ° C for 4 minutes; Reverse Transcription 60 ° C for 24 minutes; 46 cycles denaturing at 94 ° C for 30 seconds, extension / annealing at 60 ° C for 30 seconds; Final extension 60 ° C for 7 minutes Retention of the post-reaction 4 ° C Amplified Product Detection A. Gel electrophoresis The presence of the amplified product was detected by gel electrophoresis as follows. The products of the reaction were fractionated using an agarose gel (100 ml of NuSieve at 3%, 0.5% of SeaChem, and 0.5 μg / ml ethidium bromide) and current buffer solution of IX TBE (0.089 M Tris, 0.089 M boric acid, 0.0025 M EDTA disodium). The electrophoresis was carried out at 100 volts for about 1.5 hour. The ethidium bromide stained bands of the DNA were visualized using UV irradiation.

B. Kinetic PCR detection In the kinetic PCR methods described above, an intercalation dye such as ethidium bromide, which fluoresces more strongly when interspersed into double-stranded DNA, is added to the PCR. The increase in double-stranded DNA during amplification is verified by measuring the fluorescence of the dye during the reaction. Because the kinetic PCR methods only measure an increase in the total amount of double-stranded DNA, the formation of the non-specific amplification product is measured independently. To measure the occurrence of the non-specific amplification resulting from the primer-independent of the amplification of the model, reactions were carried out without the model nucleic acid. In such free reactions of the model, any increase in double-stranded DNA is attributable to the formation of the non-specific amplification product, independent of the model. The conditions of the kinetic PCR reaction were as described above, except that the ethidium bromide was added to the reaction mixture at a concentration of 1 (g / ml). The reactions were verified by measuring the fluorescence of the reaction mixture as described in EP 640 828. The fluorescence measurements were normalized by dividing an initial measurement of the fluorescence obtained during an initial cycle in the reaction although the measurements of the Fluorescence between cycles were relatively constant. The number of cycles chosen for the initial measurement of the fluorescence was the same for all the reactions compared, so that all the measurements represent increments in relation to the same cycle of the reaction. The fluorescence of the reaction in the free targets of the target or target remained relatively constant until the primer-dimer was formed. In most reactions, if sufficient amplification cycles are carried out, the dimer primer eventually becomes detectable. The effect of the modified primers can be seen from a comparison of the number of cycles carried out up to qe the primer-dimer is formed, if there is one in its entirety.

Results The results of the electrophoretic analysis of the gel are shown in Figure 3. The numbers of the strips 0 intervals corresponding to each of the amplifications using the unmodified primers and the modified primers of the three types and 200 copies, 20 copies, or 0 copies of the VCH RNA are shown in the following table (the numbers of the strips or intervals are counted from the left to the right: the bands or intervals 1-30 are in the upper half of the gel, the bands or intervals 31-60 are in the lower half of the gel). In addition, the molecular weight markers were present in the strips or intervals 1 and 31 (Hac III digested PhiX 174 RF DNA, New England Biolabs, Beverly, MA) and in strips or intervals 30 and 60 (Superladder-low, 20 bp ladder, Gen Sura, Del Mar, CA). The bands corresponding to the specific product proposed are indicated in the figure by an arrow (~ 230 bp). The other bands in the gel correspond to the non-specific amplification product and, in particular, to the primer-dimer.

Girdle Numbers or intervals of the Results of the / Amplifications Shown > s in Figure 3 Models Primers Shape or Interval 200 unmodified 2-5 200 methylated 6-9 200 benzylated 10-13 200 nitrobenzylated 14-17 20 unmodified 18-21 20 methylated 22-25 20 benzylated 26-29 20 nitrobenzylated 32-35 0 unmodified 36-41 0 methylated 42-47 0 benzylated 48-53 0 nitrobenzylated 54-59 The results demonstrate that the amplification using the modified primers led to a greater amount of the amplified HCV nucleic acid than the amplifications using the unmodified primers. In addition, the amplification used by the modified primers led to a reduction in the primer-dimer in relation to the amplifications using the unmodified primers. In the kinetic PCR evaluations, fluorescence was verified throughout the reaction. The rate of increase in fluorescence after the increase in fluorescence was detectable, was approximately the same in all reactions, as is evident from the shape of the curve obtained by plotting the fluorescence against the number of cycles (not shown). This indicated that the modified primers do not detectably inhibit the efficiency of each step of the amplification after the initial stage of amplification. The reactions were significantly different in the number of cycles carried out before an increase in fluorescence was detectable. To quantify the differences between the reactions, the results are expressed in terms of the number of amplification cycles carried out until the fluorescence exceeded a level of arbitary fluorescence (AFL). The AFL was chosen close to the fluorescence level of the baseline, but above the random fluctuations in the measured fluorescence, so that the kinetic characteristics of the reaction were measured during the geometric growth phase of the amplification. The accumulation of the amplified product in the last cycles inhibits the reaction and eventually leads to a plateau or maximum value of the reaction. The results of the kinetic PCR are summarized in the following table. Each value for the amplifications of 20 or 200 copies of the target or target model represents an average of five replicate amplifications, with the exception of the amplifications using the benzylated primers and 20 copies of the target or target, which represent an average of four. replicas or replicates. Each value for the amplifications without the model represents an average of eight replicates or replicates. Two of the eight replicates or replications of the amplifications using the benzylated primers without a target present, did not lead to the formation of the ccbador-dímcro towards the end of the 46 cycles. The average of the remaining six amplifications is shown, which represents an average of the conditions on the primer-dimer that is formed. The conditional average is not comparable with other values shown because of the deleted data.

Cycles to reach the Number of Copies of AFL White Primer 0 20 200 unmodified 35 36 34 methyl 39 38 36 riitrobenzium 43 40 37 benzyl (43 *) 41 37 * 2/8 did not show the formation of the dimer primer The data indicate that the modified primers apparently retard the amplification of target or target nucleic acid such that the AFL is reached several cycles later. The delay did not correspond to a reduction in the final yield of the specific amplification product. All amplifications of the target or target nucleic acid were observed to reach a plateau or maximum value within the 46 cycles used in the experiment and, as is evident from the corresponding data from the gel eiectrofresis analysis, the final yield was increased using the modified primers. The data indicate that the delay in primer-dimer formation was significantly greater than the delay in target or target amplification. The beneficial effect of the primers is observed more clearly comparing the free amplifications of the target or target and the amplifications of 200 copies of the model. Using unmodified primers, the increase in fluorescence with respect to AFL occurred only one cycle later in the amplifications without the target or target, indicating that the target or target amplification would be difficult to distinguish from the formation of the primer. -dimer In contrast, using the modified primers, the increase in fluorescence due to the primer-dimer occurred at least three cycles later and, using the benzylated primers, occurred at least 6 cycles later, if at all. Accordingly, target or target amplification could be detected and distinguished from primer-dimer formation. By comparing the free amplifications of the target or targets and the amplifications of 20 copies of the model, the effect of the modified primers showed the same configuration of a larger delay at the start of the primer-dimer than the delay in the target or target amplification. Using unmodified primers, 20 copies of the model could not be detected. Using the nitrobenzyl and benzyl primers, formation of the sc dimer primer delayed enough to make it possible to detect 20 copies of the model in this system.

The fluorescence verification data in each amplification cycle (data not shown), indicated that, in general, the delay in the formation of the dimer primer was sufficient to prevent the formation of the dimer primer after reaching a maximum level or of plateau within 46 cycles. Accordingly, the modified primers appear to retard the formation of the dimer primer in a sufficient manner such that the amplification of the target or target can be completed and the reaction stopped before a significant level of the dimer primer is formed.

Example 8 Photo-Labile Primers To demonstrate the use of the photolabile modified primers, amplifications of the VCH RNA were carried out using both modified primers and unmodified primers. The modified primers were modified by the attachment of one or two nitrobenzyl groups to the exocyclic aa of the 3 'terminal adenine.

Amplification primers The primers were synthesized as described in Example 4. The designations for the primers used are shown below.

Primer Seq Id.No Modification of the Base 3 ' ST280A 3 unmodified 15239 3 bis-nitrobenzyl 15241 3 mononitrobenzyl ST778AA 4 unmodified 15240 4 bis-nitrobenzyl 15242 4 mononitrobenzyl Amplification Reactions For each pair of primers, the reactions were carried out using a series of dilutions of target concentration or target entry. Two panels of the reactions, each including all the combinations of the pair of primers and the concentration of target or entry target, were carried out, and within each reaction panel, each reaction containing a given pair of primers and the concentration of target or target, were carried out in duplicate. The amplifications were carried out in 100 μl reactions containing the following reagents: 0, 10, 102, 103, 104, or 105 copies of the 55 mM VCH RNA model of Tricine, 90 mM KOAc; 3 mM Mn (OAc) 2; 200 μM each of dATP, dCTP, dGTP, dTTP, 200 μM dUTP; 250 nM of each primer; 10 U rTth *; 2U UNG *; and 8% Glycerol. * manufactured and developed by Hoffmann-La Roche and marketed by Perkin Elmer, Norwalk, CT.

The thermal cyclization of each mixture was the reaction carried out in a GeneAmp PCR System 9600 thermal cyclization device (Perkin Elmer, Norwalk, CT) using the following temperature profile: Incubation of the Pre-reaction 50 ° C for 5 minutes Reverse Transcription 60 ° C for 30 minutes; Initial Denaturation 95 ° C for 1 minute; 2 cycles: denaturation at 95 ° C for 15 seconds, annealing / extension at 60 ° C for 20 seconds; 46 cycles: denaturation at 90 ° C for 15 seconds, annealing / extension at 60 ° C for 20 seconds; Final extension 72 ° C for 10 minutes, Polished reaction tube caps (Perkin Elmer, Norwalk, CT) were used throughout the experiment. After the reaction temperature was raised to 60 ° C during the reverse transcription step, the hot shell was removed from the PCR tray in the thermal cyclerization block, and half of the reaction tubes ( a complete set of duplicate reactions) was covered with an aluminum foil. The other half was illuminated using a manually held UV lamp that emits 302 nm (UVP model UVM-57, UVP Products, San Gabriel, CA) for 10 minutes. The hot cover was replaced and amplification was continued.

Results The results of the amplifications were analyzed by gel electrophoresis as described above. The results are shown in Figure 4. The primers and the number of copies of the model used in each reaction are indicated on the gel (log of the number of copies shown). The bands corresponding to the proposed product are indicated in the figure. The other bands cn cl gcl correspond to the non-specific amplification product and, in particular, to the dimer primer. A comparison of the irradiated set with UV light of the reactions shows that the use of the modified primers led to a significant decrease in the dimer-primer, especially in the low number of copies. A comparison of the non-irradiated set of the reactions shows that the use of the bis-nitrobenzyl primers led to a complete inhibition of the amplification, as expected. Amplifications using mononitrobenzyl primers do not only produced the product, but exhibited a significant reduction in the dimer primer, which is consistent with the results obtained in the previous example.

Example 9 Amplifications using Modified Primers with p-tert-butylbenzyl This example describes the amplification of RNA VCH using the primers modified with the p-tert-butylbenzyl groups.

Nucleic Acid of Target or Target VCH RNA models were synthesized using the VCH RNA transcription vector as described in Young et al., 1993, J. Clin. Mycrobiol. 31 (4): 882-886.

Primers The amplifications were carried out using the modified primers synthesized as described in Example 2, above. The nucleotide sequences of the unmodified primers are shown subsequently, oriented in the 5 'to 3' direction. The primers used were modified versions of the upstream primer ST280A (SEQ ID NO: 3) and the downstream primer ST778AA (SEQ ID N0: 4). The modified forms of the primers are designated here as follows: Modified VCH Amplification Primers Primer Seq Id.No. Position of Modified Nucleotide ST280ATBU 3 Extreme 3 'ST778AATBU 4 Extreme 3' Amplification and Analysis The amplifications were carried out in 100 μl reactions containing the following reagents: , 5, 2.5, 2, or 0 copies of the 50 mM VCH RNA model of Tricine, (pH 8.33) 110 mM KOAc; 300 μM each of dATP, dCTP, dGTP; 50 μM dTTP; 500 μM dUTP; 50 nM of each primer; 3.5 nM of Mn (OAc) 2, 13% Glycerol. 20 units of DNA polymerase rTth, and 8.0 units of UNG *. * manufactured and developed by Hoffmann-La Roche and marketed by Perkin Elmer, Norwalk, CT.

The cyclization of the amplification temperature was carried out in a thermal cyclisation device of the TC480 DNA (Perkin Elmer, Norwalk, CT) using the following temperature profile: Incubation of the pre-reaction 45 ° C for 12 minutes; Inactivation of UNG 90 ° C for 30 seconds; Reverse Transcription 60 ° C for 20 minutes; 47 cycles: denaturation at 94 ° C for 45 seconds, annealing / extension at 60 ° C for 70 seconds; Final extension 60 ° C for 7 minutes; Retention of the post-reaction 10 ° C until analysis (for a short time) .

The products of the amplification were analyzed by gel electrophoresis, as described above.

Results The amplifications carried out in each target model number or target were replicated as follows: 3 amplifications were carried out using 20 copis of the target or target model, 3 amplifications were carried out using 5 couplets of the target or target model , 2 amplifications were carried out using 2.5 copies of the target or target model, 1 sc amplification was carried out using 2 copies of the target or target model, and 23 amplifications were carried out without any target or target present. All the positive amplifications of the model led to a single band on the gel of the expected size of the target or target. None of the amplifications led to any primer-dimer or other non-specific binding product. The results can be compared with those in Example 6, above, where the same target or VCH target was amplified using the same primer sequences. A comparison of these results with those in Example 6 indicates that the amplifications using the primers modified with p-tert-butylbenzyl were significantly improved in relation to the corresponding amplifications carried out with the unmodified primers. Additional experiments were carried out in which the HIV-1 RNA was amplified using the modified versions of p-tert-butylbenzyl of the primers described in Example 5, above. The amplifications were carried out essentially as described above. As with the VCH system described here, all positive amplifications of the HIV-1 model led to a single band on the gel of the target size or target expected, and none of the amplifications led to any primer-dimer or other non-specific amplification product. These additional results can be compared to those in Example 5, above, where the same target or HIV target was amplified using the same primer sequences. A comparison of these results with those in Example 5, above, indicate that the amplifications using the primers modified with p-tert-butylbenzyl were significantly improved with relation to the corresponding amplifications carried out with the unmodified primers.

Example 10 Amplification of Mycobacterial DNA This example describes a comparison of mycobacterial DNA amplifications carried out using modified and unmodified primers. Both the primers modified by the addition of a benzyl group to the 3 'terminal nucleotide and the primers tyiodified by the addition of a group of p-tert-butylbenzyl to the 3'-terminal nucleotide were used. The reactions using the unmodified primers were essentially as described in Tevere et al, 1996, J. Clin. Microbiol. 34 (4): 918-923. The amplifications were carried out using saliva samples within which the mycobacteria had been added at a known concentration to minimize the infected clinical samples. Additional sc amplifications were carried out using the purified microbacterial DNA, and using the negative control samples free of DNA.

Preparation of the Sample Saliva specimens that previously showed that they will be negative for mycobacteria by microscopy and culture were liquefied and decontaminated by the N-acetyl-cysteine-NaOH method by the CDC (Kent and Kubica, 1985, Public Health Mycobacteriology - a guide for the level III laboratory, US Department of Health and Human Services, Centers for Disease Control, Atlanta, incorporated herein for reference). Liquefied saliva (100 μl) was added to 500 μl of the Respiratory Specimen Wash Reagent (10 mM Tris-HCl, 1 mm EDTA, 1% (v / v) Triton X-100, 0.05% NaN3, pH 8.0) and centrifuged for 10 minutes at 12,500 x g. Each pill was resuspended in 100 μl of the lysis reagent (0.05 N NaOH, 1% (v / v) Triton X-100, i mM EDTA, 0.05% NaN3) and incubated for 45 minutes at 60 ° C. The lysates were then neutralized with 100 μl of neutralization reagent (0.2 M Tris-HCl, 8 mM MgCl 2, 0.05% NaN 3, pH 7.5). The grouped lysates of the saliva were generated by combining 80 μl each of two separate saliva lysates. To each of the 8 salivary iisatos grouped (160 μi each) was added 15 μl of a DNA storage material (2 copies / μl in a 1: 1 mixture of the lysis reagents and neutralization) purified from cultured M. tuberculosis. The samples containing the purified mycobacterial DNA (without saliva) at a known concentration were prepared by adding 10 μl of the DNA storage material to 100 μl of a 1: 1 mixture of the lysis reagent and the neutralization reagent. The negative control samples (not the DNA) consisted of a mixture of 100 μl of the lysis reagent and 100 μl of the neutralization reagent.

Amplification primers The amplifications were carried out using the primers consisting of the following nucleotide sequences: Sequence primers KY18 (SEQ ID NO: 5) 5'-CACATGCAAGTCGAACGGAAAGG-3 ' KY436 (SEQ ID NO: 6) S'-TAACACATGCAAGTCGAACGGAAA-'S 'KY75 (SEQ ID NO: 7) 5'-GCCCGTATCGCCCGCACGCTCACA-3' The following pairs of primers, which contain the indicated modifier group, fixed to the 3 'terminal base, were used in the amplifications. All the modified primers were synthesized as described in the previous examples. All primers were biotinylated at the 5 'end.

Primer Pair Primer Sequences Modification A KY18 (SEQ ID NO: 5) unmodified KY75 (SEQ ID NO: 7) unmodified B KY436 (SEQ ID NO: 6) benzyl KY75 (SEQ ID NO: 7) benzyl C KY436 (SEQ ID NO: 6) p-tert-butylbenzyl KY75 (SEQ ID NO: 7) p-tert-butylbenzyl Amplification For each sample, the amplifications were carried out using the pair of the unmodified primers, KY18 (SEQ ID NO: 5) and KY75 (SEQ ID NO: 7), and the modified forms of the pair of primers, KY436 (SEQ ID NO: 6) and KY75 (SEQ ID NO: 7). The amplifications were carried out in 100 μl reactions, each containing 50 μl of one of the three samples described above and 50 μl of a 2X reagent mixture, which contains the following reagents: 100 mM Tris-HCl, pH 8.9; 500 nM of each primer; 200 μM (each) of dNTP (dATP, dCTP, dGTP, dUTP); 20% (v / v) glycerol; 10 units of AmpliTaq; 6 units of Am? Erase (*. ^ Manufactured and developed by Hoffmann-La Roche and marketed by Perkin Elmcr (Norwalk, CT).

The thermal cyclization of each reaction was carried out in a Gene Amp PCR System 9600 thermal cyclization apparatus (Perkin Elmer, Norwalk, CT) using the following temperature profile: Pre-reaction incubation 50 ° C for 5 minutes; 2 cycles: denaturation at 98 ° C for 20 seconds, annealing at 62 ° C for 20 seconds, extension at 72 ° C for 45 seconds; 41 cycles: denaturation at 94 ° C for 20 seconds, 25 annealing at 62 ° C for 20 seconds, extension at 72 ° C for 45 seconds; Final extension 72 ° C for approximately 12 hours (overnight).

The products of the amplification were visualized by electrophoresis through 2% Nusieve®, 0.5% agarose gel followed by staining of ethidium bromide.

Results The results of the electrophoretic analysis are shown in Figure 5. For each sample, the products of the amplifications were carried out with the unmodified primers (indicated as "A") and with modified primers (indicated as "B" and "C"). ") were made to run or work on adjacent strips or intervals. The bands corresponding to the proposed target or mycobacterial target sequence are indicated by arrows. Other bands correspond to the non-specific amplification product; the lower bands in the gel correspond to the primer-dimer. Strips or ranges marked "M" contain a molecular weight marker (Hae III digestion of PhiX174 DNA).

Using the unmodified primers, the amplifications of the purified mycobacterial DNA led to the formation of the primer-dimer. The use of any of the modified pairs increased the amount of the proposed target present and eliminated that cial-neite formation of the de-tectabie-diter primer. In contrast to the amplifications of the purified DNA, using the unmodified primers, the presence of salivary lysate in the amplification reaction reduced the efficiency and increased the formation of the non-specific amplification product, as reported by the presence of the foreign product bands The increase of the non-specific amplification product is not surprising given that the saliva lysates contain a significant amount of human DNA, which is not present in the amplifications of the purified mycobacterial DNA. any of the primer pairs modified in the amplifications carried out in the presence of the saliva, led "both to a significant increase in the amount of the proposed product generated as well as to a reduction of the non-specific amplification.

Example 11 Additional Synthesis of Modified Primers with a Benzyl Group Primers modified by the addition of a benzyl group to the terminal cytosine were synthesized essentially as described in Example 1, but using an N-acetyl, N-benzyl-5-o-DMT-2-deoxycytidine bound or bound to LCAA-CPG, prepared as described below.

Step 1: Synthesis of N4-benzyl-2-deoxycytidine To 2? -deoxycytidine hydrochloride (5.28 g, 20 mmol, U.S. Biochemical Corp., Cleveland, OH) benzylamine (20 ml) was added, and the mixture was heated at 150 ° C for 3 hours under an argon atmosphere. The solution was concentrated under vacuum to give a viscous yellow oil, which was partitioned between water (100 ml and ethyl acetate (100 ml), the aqueous phase was washed with ethyl acetate (100 ml) and separated. The mixture is concentrated under vacuum to give a yellow syrup (13 g), which is purified by column chromatography on silica gel with methylene chloride: methanol 15: 1 as -J ^ _ ^ eluent, to give the desired product (5.8 g, 91.5%), * as a colorless syrup.

Step 2: Synthesis of N-acetyl, N4-benzyl-2 - 5 deoxycytidine The N 4 -benzyl-2α-deoxycytidine (2.5 g, 7.9 mmol) is dissolved in 15 ml of dry dimethylformamide (15 ml). • e ml), acetic anhydride (8 g, 79 mmol, 10 g) is added. eq.), And the mixture is stirred overnight at room temperature. The solvent and excess acetic anhydride were evaporated under vacuum. The product was purified by column chromatography with silica gel using methylene chloride: methanol 20: 1 as the eluent, give the title compound (1.3 g, 48%). The compound was highly hygroscopic and was stored desiccated at -20 * ° C.

Step 3: Synthesis of N4-acetyl, N4-benzyl, 5-20 O-DMT-2 '-deoxycytidine.

N4-acetyl, N4-benzyl-2'-deoxycytidine (76 mg, 0.2 mmol) was dissolved in 1 ml of dry pyridine, and DMT-C1 (122 mg, 0.2 mmol, 1.0 eq.) Was added. Mix of the reaction is stirred for 3 hours. The CCD analysis showed that some of the match material was left, so that an additional aliquot of DMT-C1 (61 mg, 0.5 eq.) was added and the resulting mixture was stirred for another hour, at which time the CCD analysis showed that the reaction was complete. The reaction was quenched with 15 ml of brine solution and the aqueous phase was extracted with pietyiene cioride (3 X 15 ml). The combined organic layers are washed with brine (2 X 15 ml) and dried over anhydrous magnesium sulfate. The solvent is evaporated and the mixture is purified by silica gel chromatography using methylene chloride: methanol 50: 1, to give N 4 -acetyl, N 4 -benzyl, 5? -0-DMT-2 '-deoxycytidine (96 mg. , 65% yield).

Step 4: Succinylation The N 4 -acetyl, N-benzyl, 5 -0-DMT-2α-deoxycytidine (96 mg, 0.13 mmole) was dissolved in 2 ml of dry pyridine. Succinic anhydride (100 g, 1.0 minol) and dimethylaminopyridine (20 g) are added, and the resulting mixture is stirred at room temperature for three days. The solvent was evaporated and the residue coevaporated with toluene (3 X 10 ml). The chloroform (50 ml) is added to dissolve the residue (the sound application is used to aid the dissolution). The chloroform layer is washed with brine (3 X 15 ml), and water (1 X 15 ml). The organic layer is dried with sulfate • anhydrous magnesium. The solvent was evaporated to give 108 mg of N4-acetyl, N4-benzyl, 5? -0-DMT-2? -deoxycytidine-3'-0-succinate (97% yield).

Step 5: Preparation of 5? -0-DMT-N4-acetyl, N4-benzyl-2-deoxycytidine-3? -0-succinate bound to the LCAA-CPG The activated CPG was prepared as follows. LCAA-10 CPG (1.0 g, LCA00500C, CPG Inc., Fairfield, NJ) was prepared with trichloroacetic acid in methylene chloride (3%, 10 ml) and mixed by rotating on a rotary evaporator (rotoevaporator, Buchi , t'lawil, Switzerland) (without vacuum) for 4 hours. The solvent was removed by filtration and the CPG was washed with triethylamine: ethyldiisopropyl ina 9: 1 (3 X 5 ml), methylene chloride (3 X 10 ml), and ether (3 X 10 ml) consecutively, then dried under vacuum. The union of the nucleoside intermediate modified with the CPG washed with acid was carried out as follows. To 1 gram of activated LCAA-CPG was added N4-acetyl, N4-benzyl, 5'-0-DMT-2-deoxycytidine-3? -0-succinate (108 mg, 0.13 mmol), prepared as described above, dimethylaminopyridine (20 mg), and 5 ml of dry pyridine. The reaction mixture was rotated on a rotoevaporator (without vacuum) for three days. The supernatant is removed by filtration, and the bound LCAA-CPG is washed sequentially with pyridine (3 X 5 ml), methylene chloride (3 X 10 ml), and ether (3 X 10 ml), and then dried under vacuum . Coronation at the ends of the LCAA-CPG unit with the N 4 -acetyl, N-benzyl, 5? -0-DMT-2? -deoxycytidine-3'-O-succinate was carried out as follows. To the derived CPG is added the coronation mixing reagent A at the ends (THF / Lutidin / Ac20 8: 1: 1, DNA synthesis reagents from Glen Research, Sterling, V) and B (10% N-methylimidazole in THF, Glen Research), and the reaction mixture was rotated on a rotoevaporator (without vacuum) all night. The solution is removed by filtration, and the bound LCAA-CPG is washed sequentially with pyridine (3 X 5 ml), methylene chloride (3 X 10 ml), THF (3 X 10 ml), and ether (3 X 10 ml). ), and then dried under vacuum. The binding capacity of the derived LCAA-CPG was determined by treating 5 mg of the product with 3% trichloroacetic acid in methylene chloride, and the amount of the dimethoxytrityl carbonium ion released is measured by UV spectroscopy. The amount of the nucleoside derivative bound to the LCAA-CPG was determined to be 19.5 μmol / g.

LIST OF THE SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: F. HOFFMANN-LA ROCHE AG (B) STREET: Grenzacherstrasse 124 (C) CITY: Basle (D) STATE: BS (E) COUNTRY : Switzerland (F) ZIP CODE: CH-4070 (G) TELEPHONE: 061 - 688 25 11 (H) TELEFAX: 061 - 688 13 95 (I) TELEX: 962292/965542 hlr ch (ii) TITLE OF THE INVENTION: Modified Primers (iii) NUMBER OF SEQUENCES: 7 (iv) READABLE FORM OF THE COMPUTER: (A) TYPE OF MEDIUM: flexible magnetic disk (B) COMPUTER: Apple Macintosh (C) OPERATING SYSTEM: System 7.1 (Macintosh) (D) PROGRAM: Word 5.1 (v) DATA OF THE PREVIOUS APPLICATION; NUMBER OF APPLICATION: 60-041127 DATE OF SUBMISSION: 20.03.97 (2) INFORMATION FOR SEQ ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: l: CAATGAGACA CCAGGAATTA GATATCAGTA CAA 33 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: CCCTAAATCA GATCCTACAT ATAAGTCATC CA 32 (2) INFORMATION FOR SEQ ID NO: 3: 5 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid '00 (C) TYPE OF HEBRA: only 10 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: GCAGAAAGCG TCTAGCCATG GCGTTA 2ß Jk? (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: 20 (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: GCAAGCACCC TATCAGGCAG TACCACAA 28 (2) INFORMATION FOR SEQ ID NO: 5: 5 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid 'J. (C) TYPE OF HEBRA: only 10 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: 15 CACATGCAAG TCGAACGGAA AGG 23 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: unique (D) TOPOLOGY: linear 25 ii) TYPE OF MOLECULE: DNA (genomic; (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: TAACACATGC AAGTCGAACG GAAA 24 (2) INFORMATION FOR SEQ ID NO: 7: 5 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucieic acid J (C) TYPE OF HEBRA: only 10 (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 15 GCCCGTATCG CCCGCACGCT CACA 24 It is noted that in relation to this date the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following

Claims (11)

1. An oligonucleotide that has the general structure: R R I I 5'-S1-N-3 'or 5'-S1-N-S2-3', characterized in that: If it represents a first nucleotide sequence of between about 5 and about 50 nucleotides in length; S2 represents a second sequence of between one and three nucieotides in length; N represents a nucleotide containing a purine base or pyrimidine containing an exocyclic amine; R represents a modifying group, R is covalently linked to N via the exocyclic amine, and R has the structure: where Ri and R > 2 independently represent hydrogen, an alkyl group with Ci-Cio, an alkoxy group, a phenyl group, a phenoxy group, a substituted phenyl group, a naphthyl group, or a substituted naphthyl group.

2. An oligonucleotide according to claim 1, characterized in that R is a 2-naphthyl ethyl group; a benzyl group; or a substituted benzyl group.

3. An oligonucleotide according to claim 1 or claim 2, characterized in that R is a substituted benzyl group having the structure: wherein R3 represents a linear or branched alkyl group with Ci-Cß, a methoxy group, or a nitro group.

4. An oligonucleotide according to any of claims 1-3, characterized in that R3 represents a linear or branched alkyl group with C? ~ C4, a methoxy group, or a nitro group. - • > 5

* 5. An oligonucleotide according to claim 3 or claim 4, characterized in that R3 is fixed in the para position.

6. An oligonucleotide according to any of claims 1-5, characterized in that N is adenosine.

7. An oligonucleotide in accordance with 10 any of claims 1-6, characterized in that R is selected from the group consisting of benzyl, p-methylbenzyl, p-tert-butylbenzyl, p-methoxybenzyl, o-nitrobenzyl, and 2-naphthymimethyl.

8. A method for amplifying a nucleic acid target or target sequence, characterized in that the method comprises carrying out an amplification reaction using at least one oligonucleotide according to any of claims 1-7.

9. A method according to claim 8, characterized in that the method is the chain reaction of the polymerase.

10. A set or set to carry out the amplification reaction of the nucleic acid, characterized in that the kit or assembly comprises an oligonucleotide according to any of claims 1-7.

11. The new oligonucleotides, methods and sets or sets, characterized in that they are substantially as described here above. 10 fifteen twenty 25