WO1993024658A1

WO1993024658A1 - Signal amplification probe and methods of use

Info

Publication number: WO1993024658A1
Application number: PCT/US1993/004950
Authority: WO
Inventors: Linxian Wu; James Michael Kadushin
Original assignee: Gen Trak, Inc.
Priority date: 1992-05-29
Filing date: 1993-05-26
Publication date: 1993-12-09
Also published as: AU4596893A

Abstract

A signal amplification probe for the detection of an immobilized target molecule in a sample. The probe contains 1) a transcription-based amplifier construct, and 2) a multivalent bridging molecule for direct or indirect attachment of the amplifier construct to the immobilized target molecule. The amplifier construct contains a polymerase promoter and a template for transcription by a polymerase that binds to the promoter. The signal amplification probe can be used in combination with either a capture probe for immobilizing the target molecule to a solid phase, a linker probe for attaching the multivalent bridging molecule to the target molecule, or both. Assays utilizing the probe and kits containing the probe are also provided.

Description

SIGNAL AMPLIFICATION PROBE AND METHODS OF USE

Background of the Invention

This relates to the field of molecular biology, and more particularly relates to molecular probes for use in biological assays.

Cross-reference to Related Applications

This is a continuation-in-part of U.S. Patent Application Serial No. 07/893,097, filed May 29, 1992 by Kadushin and Mowshowitz, entitled "Nucleic Acid Constructs Useful as Signal Amplifiers".

Molecular probes are well known biochemical tools used by scientists and clinicians to detect molecules of cellular and viral origin. Generally, a probe specific for the molecule being detected is combined with a biological sample, and the probe becomes attached to the target molecule and generates a detectable signal. Subsequent detection of the signal indicates the presence of the target molecule and can indicate the relative amount of target in the sample.

Successful binding of the probe to the target molecule often depends on the type of target molecule being detected. For example, a probe for a nucleic acid target molecule is usually a nucleic acid molecule that binds to the target by hybridization of the complementary portions of each molecule. Alternatively, a probe specific for a protein often binds through an antibody molecule specific for an epitope on the protein. The detectable portion of the bound probe has traditionally been a substance chemically affixed to the probe such as a chemical label. Examples of chemical labels are radioisotopes, substrate- modifying enzymes such as alkaline phosphatase, enzymatic cascade processes utilizing NADP, and fluorescent compounds. Chemical labels have been used in immunoassays, nucleic acid assays, and other biological assays with great success. However, chemical labels are often not sensitive enough to detect very low levels of target-bound probe, particularly in assays where there is significant background noise. Assays utilizing nucleic acid probes, to which a chemical label has been attached, are described in U.S. Patent No.

4,851,331 to Vary et al.; U.S. Patent No. 4,581,333 to Kourilsky et al.; U.S. Patent No. 4,994,373 to Stavrianopoulos et al.; U.S. Patent No. 4,882,269 to Schneider et al.; and International Patent Application Publication No. WO87/03622 to Princeton Univ.

Recent probe technology has utilized signal- amplification systems that amplify the signal for detection of smaller quantities of target molecule. For example, in nucleic acid assays, the polymerase chain reaction (PCR) and similar processes are used to increase the number of copies of the target molecule to which the detectable probe will bind. PCR has emerged as a powerful and sensitive procedure for the amplification of specific DNA sequences, and is a valuable tool in paternity identification, tissue typing, and forensics. PCR technology requires pairs of dissimilar DNA oligonucleotides that act as primers to initiate a controlled polymerase reaction for amplification of the genomic sequence that lies between the two oligonucleotide binding sites. The polymerase chain reaction employs the heat-stable Taq polymerase that permits repeated heating and cooling of the reaction mixture. The amplification process is initiated by first heating the reaction mixture to denature, or dissociate, the two complementary strands of the double-stranded DNA to be amplified. Upon cooling, each single-stranded DNA oligonucleotide hybridizes to a specific region of one or the other of the complementary DNA strands and acts as a primer for the heat-stable polymerase. The polymerase uses the oligonucleotide primers as starting points for the elongation of a DNA molecule complementary to the template..DNA molecule to which each primer is hybridized. Each of the elongating DNA chains grows towards and beyond the distal primer site of the other template strand. By the end of the first cycle, two double-stranded copies of the intervening genomic sequence lying between the primer binding sites are generated. The cycle is repeated many times, exponentially doubling the number of copies each time. In this way, even a single copy of a specific DNA sequence can be amplified to detectable levels in a relatively short period of time.

PCR technology is described in U.S. Patent No. 4,683,202 to Mullis; U.S. Patent Nos. 4,683,195 and 4,965,188 to Mullis et al.; and International Patent Application Publication No. 092/14843 to Gilead Sciences Inc.

Other assays employing the concept of signal amplification utilize DNA-dependent RNA polymerases for the amplification of portions of either the target molecules or probe molecules bound to target molecules. In this type of signal amplification assay, an RNA polymerase promoter sequence is included as part of a single-stranded DNA probe so that, after hybridization of the probe to the target, the promoter can be used to promote polymerase-catalyzed synthesis of target or probe segments. Assays employing DNA transcription-based amplification systems are described by Kwoh et al.. Proc. Natl . Acad. Sci . USA 86:1173-1177 (1989); Guarelli et al., Proc. Natl . Acad. Sci . USA 87:1874-1878 (1990); Japanese Patent Application No. 2,131,599 to Toray Ind. Inc.; International Patent Application Publication No. W091/17442 to Chiron Corp. ; International Patent Application Publication No. WO91/10746 to Chiron Corp.; and International Patent Application Publication No. WO89/06700 to Genentech, Inc. In a similar RNA based amplification system, a nucleic acid probe containing an RNA molecule is autocatalytically replicated using Qβ bacteriophage replicase as described by Chu et al., Nύcl . Acids Res . 14:5591-5603 (1986); U.S. Patent No. 4,957,858 to Chu et al.; U.S. Patent No. 4,786,600 to Kramer et al.; and International Patent Application No. 092/12261 to Promega Corp.

The main disadvantage to the presently available molecular probes is that the signal amplification efficiency is only a 10 to 20 fold increase over conventional hybridization technology such as the ImmmunoSelect™ ELISA Amplification System (GIBCO BRL, Gaithersburg, MD) .

In addition, two drawbacks are commonly related to the powerful amplification technologies currently available, namely, contamination and difficulty in quantitation. In order to overcome these two problems, The Perkin-Elmer Corporation, (Norwalk, CT) has developed the UNG-contamination™ Prevention Kit to solve the contamination problem, and Clontech Laboratories Inc. (Palo Alto, CA) has introduced COMPETITIVE PCR™ to quantitate PCR products. The disadvantages to both of these kits are that they require additional steps, are time consuming and are costly.

Therefore, it is an object of the present invention to provide a probe having high sensitivity and specificity for detection of low concentrations of a target molecule.

It is a further object of the present invention to provide a signal amplification probe having a high amplification efficiency.

It is a further object of the present invention to provide a probe that can be used to quantitate the amount of target molecule in a sample. It is a further object of the present invention to provide an assay for the detection of a target molecule that is simple, rapid, and cost- efficient.

Summary of the Invention

A signal amplification probe for the detection of a target molecule, such as a protein or nucleic acid molecule, in a biological sample is provided. Detection of a specific nucleic acid molecule is particularly useful for tissue typing or paternity testing. Assays utilizing the probe and kits containing the probe are also provided.

The signal amplification probe contains 1) a transcription-based amplifier construct, and 2) a multivalent bridging molecule for direct or indirect attachment of the amplifier construct to an immobilized target molecule. Preferably, the amplifier construct is composed of two partially overlapping nucleic acid molecules forming a double-stranded portion and a single-stranded portion. The double-stranded portion provides an RNA polymerase promoter while the single-stranded portion serves as a template for transcription by a polymerase that binds to the promoter. The production of multiple, detectable RNA molecules as a result of the transcription of a single amplifier construct provides a large portion of the desired amplification effect.

In a first preferred embodiment, the signal amplification probe is used in combination with a capture probe for immobilizing the target molecule to a solid phase. In a second preferred embodiment, the target molecule is bound to a solid phase sphere via a receptor-ligand interaction, and the signal amplification probe is used in combination with a linker probe for attaching the bridging molecule to the target molecule. In a third preferred embodiment, the signal amplification probe is used in combination with both a capture probe for immobilizing the target molecule to a solid phase and a linker probe for attaching the bridging molecule to the target molecule.

The immobilized target molecule/amplifier construct complex is detected by transcription of the amplifier construct with an appropriate polymerase in the presence of multiple indicator molecules that become incorporated into each transcript. Transcripts containing the indicator molecules are transferred to a solid phase vessel coated with a probe specific for the transcripts, and the detector molecules are detected by standard methods known to those skilled in the art.

Brief Description of the Drawings

Fig. 1 is a schematic representation of biotinylated amplifier construct molecules bound to streptavidin bridging molecules that are bound to biotinylated DNA target molecules indirectly immobilized on the surface of a solid phase vessel.

Fig. 2 is a schematic representation of biotinylated amplifier construct molecules bound to streptavidin bridging molecules that are indirectly bound to biotinylated DNA target molecules directly immobilized on the surface of a solid phase bead. Figure 3 is a schematic representation of biotinylated amplifier construct molecules bound to streptavidin bridging molecules that are indirectly bound to DNA target molecules indirectly immobilized on the surface of a solid phase bead. Figure 4 is a bar graph showing the optical density of labelled RNA transcripts generated by biotinylated and non-biotinylated amplifier construct molecules bound to biotinylated DNA target molecules immobilized on the surface of a solid phase vessel. The criss-crossed bars represent the optical density generated when biotinylated amplifier construct molecules are bound to a streptavidin bridging molecule that is bound to an immobilized biotinylated DNA target molecule. The striped bars slanting downward from left to right represent the optical density generated by non-biotinylated amplifier construct molecules. The striped bars slanting upward from left to right represent the optical density generated by biotinylated amplifier construct molecules bound to a streptavidin-alkaline phosphatase bridging molecule to block streptavidin and biotin interaction. A 1 μl aliquot of 9020* contains 2.5 x 10⁹ target molecules. The label "no DNA" is an abbreviation for "no target DNA molecules".

Figure 5 is a graph showing the optical density of a 10 μl aliquot of a 50 μl total volume of labelled RNA transcripts generated by biotinylated amplifier construct molecules bound to various concentrations of biotinylated DNA target molecules (9020*) immobilized on the surface of a solid phase vessel in the presence of 1 x 10¹³ non- target DNA molecules (fragment size = 60 bases) . A 1 μl aliquot of 9020* contains 2.5 x 10⁹ target DNA molecules.

Figure 6 is a bar graph showing optical density of 10 μl from a total volume of 50 μl labelled RNA transcripts generated as described in Figure 5.

Figure 7 is a bar graph showing optical density of labelled RNA transcripts generated by biotinylated amplifier construct molecules bound to biotinylated DNA target molecules immobilized on solid phase beads in the presence of non-target synthetic biotinylated oligonucleotides. The bar labelled "AC*/BL10UG" represents the optical density of transcripts generated from a sample containing 10 μg of synthetic biotinylated oligonucleotides to block the binding of the biotinylated amplifier construct to the streptavidin bridging molecule. The bar labelled "AC*" represents the optical density of transcripts generated from a sample containing the biotinylated amplifier construct. The bar labelled "AC/BL10UG" represents the optical density of transcripts generated from a sample containing 10 μg of synthetic biotinylated oligonucleotides to block the binding of the non-biotinylated amplifier construct to the streptavidin bridging molecule. The bar labelled "AC" represents the optical density of transcripts generated from a sample containing a non-biotinylated amplifier construct. The bar labelled "BL-only" represents the optical density of transcripts generated from a sample containing 10 μg of the synthetic biotinylated oligonucleotides in the absence of biotinylated amplifier construct. The bar labelled "NO-AC/BL" represents the optical density of transcripts generated from a sample containing no amplifier construct and no synthetic biotinylated oligonucleotide blocking molecules.

Figure 8 is a graph showing the optical densities of labelled RNA transcripts generated by biotinylated amplifier construct molecules bound to various concentrations of biotinylated DNA target molecules (9020*) immobilized on solid phase beads divided by the optical density generated by biotinylated amplifier construct molecules in the absence of target molecules. The average of ratios from four separate experiments is shown.

Detailed Description of the Invention

A signal amplification probe for the detection of a target molecule in a biological sample is described. Preferably, the target molecule is a biological molecule such as a protein, nucleic acid molecule, or sugar moiety. The target molecule is most preferably immobilized on a solid phase.

Detection of such a molecule is particularly useful for tissue typing or paternity testing. Assays utilizing the probe and kits containing the probe are also described. The signal amplification probe contains 1) a transcription-based amplifier construct, and 2) a multivalent bridging molecule for direct or indirect attachment of the amplifier construct to the target molecule. Transcription-Based Amplifier Construct

The amplifier construct is a nucleic acid molecule capable of transcription or replication. The nucleic acid molecule can be partially or fully double-stranded RNA, partially or fully double- stranded DNA, single-stranded RNA or single- stranded DNA. The double-stranded nucleic acid molecule is transcribed by the binding of a polymerase to a promoter. A useful polymerase for transcription of double-stranded RNA is Reovirus RNA polymerase. A single-stranded RNA molecule is transcribed by the binding of a polymerase to a promoter or the binding of a replicase to a replicase binding site. An example of a replicase that functions with a single-stranded RNA binding site is Qβ replicase.

An amplifier construct composed of only one nucleic acid strand ("single-stranded constructs") can be made using the techniques described below for preparation of the double-stranded constructs by merely omitting the second strand. The single- stranded constructs can be used in any of the processes or kits of the invention described below in place of the amplifier constructs with double- stranded nucleic acid.

Preferably, the amplifier construct is composed of a double-stranded DNA molecule. Most preferably, the amplifier construct is composed of two partially hybridizing DNA molecules and includes three components: a) an RNA polymerase promoter region to which an RNA polymerase molecule will bind, b) a ligand capable of binding to the multivalent bridging molecule, and c) a region capable of being transcribed. Promoter Region

Each nucleic acid molecule of the amplifier construct has a segment with a sequence that is complementary to a segment of the other nucleic acid molecule so that the complementary sequences hybridize to one another, under standard hybridization conditions, to form a double-stranded portion flanked by two single-stranded portions, as shown in Figures 1-3. The double-stranded portion of the amplifier construct forms the RNA polymerase promoter. Preferably, the complementary sequences are between approximately 15 and 20 nucleotide bases in length. The 5 ' end of one of the single- stranded portions contains the ligand molecule, and the 5' end of the second single-stranded portion serves as a template for transcription by a polymerase that binds to the promoter. The production of multiple detectable RNA molecules as a result of the transcription of a single amplifier construct provides the desired amplification effect.

In a preferred embodiment, one strand of the amplifier construct, denoted here as the "minus" strand, contains both the RNA polymerase binding sequence for the promoter region of an RNA polymerase and a region serving as the template for transcription by an RNA polymerase bound to that promoter. The "plus" strand of the construct contains regions complementary to both the promoter region of the minus strand and the region serving as a template for transcription. Within the minus strand, the template sequence is positioned so that it is "downstream" from the promoter. The promoter has two nucleotide sequences, one on each nucleic acid strand. After a polymerase binds to the promoter it will move in the 3' direction with respect to the minus strand. For each promoter, it is known in the art which of the two strands is the minus strand.

Within the minus strand, the 3' end of the RNA polymerase binding sequence is either at the 3' end of the strand or is separated from that 3' end by a "leader" sequence. Particularly when a ligand is linked to the 5' end of the plus strand, it is preferable that there is a double-stranded leader region of at least about 25 base pairs in length so that the binding efficiency of the RNA polymerase to the promoter will not be decreased by the presence of the ligand.

The stability of the double-stranded region containing the promoter is dependent upon its length and GC content. The longer the region and the greater the GC content, the more stable the double-stranded region will be when challenged with high temperature and low salt concentration under assay conditions. It will be understood by one skilled in the art that one may delay bringing the complementary strands of the amplifier construct together until after the strand with the ligand has reacted with the multivalent bridging molecule but before the RNA polymerase molecule has been added.

It will be further understood that each nucleic acid molecule of the amplifier construct can be either DNA or RNA. Preferably, both nucleic acid molecules are DNA molecules. Amplifier Construct Ligand

The amplifier construct has four strand ends (the 3' and 5' ends of the plus and minus strands) and may have a ligand covalently linked to one, two, three or all of those ends. Preferably, the ligand of the amplifier construct is linked to the 5' end of the plus strand as shown in Figures 1-3 It will also be understood by one skilled in the art that the ligand of the amplifier construct can be covalently linked to the nucleic acid molecule either directly or by means of a cross- linker molecule. The ligand of the amplifier construct is chosen so that it will bind the amplifier construct to the multivalent bridging molecule. The ligand can bind directly to the multivalent bridging molecule or indirectly through an intermediate molecule such as a probe or antibody. Preferably, the ligand of the amplifier construct binds directly to one of the receptor sites of the multivalent bridging molecule and is the same as the ligand binding the target molecule to the multivalent bridging molecule, as described in more detail below, so that only one multivalent bridging molecule lies between the amplifier construct and the target molecule.

Suitable ligands include any ligands that bind to a binding site on a multivalent receptor molecule, such as a biotin molecule that binds to streptavidin or avidin, a metal or metal ion that binds to a chelating agent, a digoxigenin molecule that binds to an anti-digoxigenin antibody (digoxigenin is also known to those skilled in the art as lanadigenin or 3B,12B,14-trihydroxycard-

20(22)-enolide) , or a carbohydrate that binds to a lectin. Most preferably, the ligand is a biotin molecule.

The ligand for the multivalent bridging molecule is covalently attached to the amplifier construct, using a cross-linker molecule, by the following procedure:

1. Treat the ligand, such as a protein, peptide, nucleic acid, carbohydrate, or the like, with a reactive heterobifunctional or homobifunctional cross-linker capable of covalently binding to an amine group or similar reaction site on the molecule. Preferably, the ligand is treated with a reactive heterobifunctional cross-linker. Typical heterobifunctional cross-linkers include SMCC (succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate) , Sulfo-SMCC (sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane- 1-carboxylate ), SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate and similar compounds; 2. remove excess non-bound cross-linker molecules by column chromatography or similar methods; and

3. add a single-stranded nucleic acid molecule, intended to be the minus DNA strand in the amplifier construct, which contains a chemical group reactive with the cross-linker, such as a thio group. Preferably, the nucleic acid molecule is a DNA molecule with a 5' thio group. Covalent binding of the 5' thio groups to the still unreacted end of the cross-linker already covalently bound to the ligand will then occur under appropriate conditions as described in The Pierce Chemical Company ImmunoTechnology Catalog and Handbook (The Pierce Chemical Company,

Rockville IL, 1990) and the book PCR Protocols - A Guide to Laboratory Technique. Gelphan, Semitssky, White. Academic Press, which are incorporated by reference herein. Subsequently, the amplifier construct is completed by hybridizing the plus DNA strand to the cross-linked minus strand. Hybridization is accomplished by combining the plus and minus strands of the construct and incubating for approximately 5 minutes at 65°C in the presence of TE buffer (BioWhittaker Inc., Walkersville, MD) , after which the incubation mixture is cooled down to room temperature and allowed to stand at room temperature for at least 15 minutes, at a concentration of not less than 1 microgram of each strand per milliliter of solution. These conditions will cause the hybridization of the plus and minus strands to each other such that nearly all of the single-stranded DNA is depleted from the reaction solution. These techniques and chemical reagents are generally available from a number of vendors, including the Pierce Chemical Co. (Rockford, IL) through their catalogs, and the references cited therein. Target Molecule

The target molecule can be virtually any molecule at all, organic or inorganic. Preferably, it is a molecule that is part of a living organism or cell (or a virus in such an organism or cell) or is a molecule that has been isolated from a living organism or cell. For example, the target molecule may be a DNA moiety comprising a gene, an RNA moiety such as an mRNA molecule, a protein- containing molecule such as a protein or glycoprotein, a hapten, or an antibody. As a result, the target molecule may also be referred to as "an analyte" or an "analyte molecule", which is the substance being determined in an analytical procedure. The terms "target molecule", "analyte", and "analyte molecule" are interchangeable as used herein. Most preferably, the target molecule is a nucleic acid molecule.

The target molecule is preferably immobilized, either directly or indirectly, to a solid phase, such as the surface of a vessel or a bead. Solid phase surfaces for immobilizing the target molecule include, but are not limited to, various membranes designed to capture nucleic acids, such as nitrocellulose and nylon membranes. Other solid phase compositions include plastic polymers and variations thereof, including chemical constructs designed to facilitate the covalent or non-covalent binding of nucleic acid to the substrate. A preferred solid phase surface is a polystyrene surface grafted with secondary amino groups which may serve as bridgeheads for further covalent binding (available from NUNC Corporation, Napier, IL and A/S NUNC, Roskilde, Denmark) . Most preferably, the solid phase is the surface of a commercially available microtiter plate well or a substrate-coated bead as described in more detail below.

For direct binding of the target molecule to the solid phase, the target molecule preferably includes a ligand that binds to a receptor attached to the solid phase. Indirect binding of the target molecule to the solid phase is preferably accomplished by hybridizing the target molecule to a complementary sequence of a capture molecule that is bound to the solid phase as described in more detail below. Multivalent Bridging Molecule

The multivalent bridging molecule connects the target molecule, either directly or indirectly, to the amplifier construct and is any molecule to which two or more ligands can bind. Preferably, the multivalent bridging molecule contains one binding site to which the target molecule is bound and multiple binding sites to which amplifier construct molecules can be bound so that further amplification of the signal generated by the transcripts is achieved.

Preferably, the multivalent bridging molecule is streptavidin, avidin or a chelating agent such as, but not limited to, methyliminodiacetic acid, which binds to divalent cations such as Mg++ or Ca++. Most preferably, the multivalent bridging molecule is streptavidin. Non-covalent binding of the amplifier construct to the multivalent bridging molecule may be accomplished by a number of methods. For example, if the multivalent bridging molecule is an avidin (or streptavidin) molecule, biotin may be used as the ligand. As biotin has a very high affinity for avidin (K disassociation 10^"15 M) , a non-covalent bond of high efficiency will be formed between the amplifier construct and the multivalent bridging molecule.

Various other binding molecules, known to those skilled in the art, can be used as the multivalent bridging molecule including, but not limited to. Protein A or Protein G, which bind to the IgG constant region; alpha-lactalbumin, which binds to galactosyl transferase; O-phosphoyl- ethanolamine, which binds to C reactive protein; polymyxin B, which binds to E. coli lipopolysaccharide; riboflavin binding protein, which binds to riboflavin; and penicilloic acid, which binds to penicillinase. Detection The target molecule-multivalent bridging molecule-amplifier construct complex is detected by transcription of the amplifier construct with an appropriate polymerase. During transcription, multiple indicator molecules, such as chemically labelled nucleoside triphosphate molecules, are incorporated into each transcript.

The RNA transcripts can be detected by a number of different methods, most of which involve their capture and then an assay for the captured RNA. Capture methods include, but are not limited to, capture by nucleic acid binding media (such as nitrocellulose or nylon) or hybridization to nucleic acid single stranded sequences of complementary base sequence. Assays can be used to detect RNA on the basis of its ability to absorb ultraviolet light, the fact that they contain radioactive markers such as ³²P, and other methods, some of which are illustrated in the Examples below. Such capture and assay techniques are well- documented in the literature.

The indicator molecules are preferably harvested and transferred to a solid phase vessel coated with a probe specific for the indicator molecules. Immobilized, labelled transcripts are then detected by standard detection methods known to those skilled in the art. Preferably, the multiple indicator molecules are detectable molecules such as, but not limited to, a biotinylated nucleotide triphosphate (ATP, CTP, GTP, or TTP) , digoxigenin-dUTP, ^-(6- aminohexyl)dATP and 5-bromo-deoxyuridine. Biotinylated nucleotide triphosphates are detected by their ability to bind to streptavidin or avidin and are available from Pierce, Rockford, IL. Digoxigenin-dUTP is capable of being bound to an anti-digoxigenin antibody and is manufactured by Boehringer-Mannheim (Indianapolis, IN). N⁶-(6- aminohexyl)dATP (manufactured by GIBCO BRL, Life Technologies, Gaithersburg, MD) , contains a primary amino group attached at the N⁶ position of the purine base by a 6 carbon linker and is therefore capable of reacting with any substrate that reacts with a primary amine. 5-bromo-deoxyuridine is a dUTP analog that may be detected by binding with an anti-bromo-dU antibody and is available from Synthecell, Inc., Rockville, MD. Most preferably, the multiple indicator molecules incorporated into the transcript are biotinylated nucleotide triphosphates. Preferred Embodiments

In a first preferred embodiment, as shown in Figure 1, the signal amplification probe is an amplifier construct 5 and multivalent bridging molecule 40 in combination with a capture probe 10 for immobilizing the target molecule 30 to the surface of a solid phase vessel 20, such as a test tube or microtiter plate well. Preferably, the target molecule 30 is a nucleic acid molecule that has been simultaneously amplified and modified to incorporate a ligand 35 for the multivalent bridging molecule 40. Most preferably, the target molecule 30 is amplified using a nucleic acid amplification system, such as PCR, in the presence of a ligand molecule that becomes incorporated at the 5' end of the target molecule.

The amplified, ligand-containing target molecule is immobilized to the surface of a solid phase vessel 20, to which has been bound a capture probe 10, such as an antibody molecule or oligonucleotide probe, by hybridizing the 3' end of the ligand-containing nucleic acid target molecule 30 to the capture probe 10. If the capture probe 10 is a nucleic acid molecule then the target molecule 30 binds to the capture probe 10 by hybridization of complementary regions of each molecule. If the capture probe 10 is an antibody, then preferably a nucleic acid molecule complementary to a "unique" or highly variable portion of the target molecule is conjugated to the antibody via covalent linking and the target molecule then binds to this conjugated nucleic acid molecule by hybridization.

The multivalent bridging molecule 40, which is attached to the amplifier construct 5 of the signal amplification probe by the interaction of a ligand 55 with a receptor, attaches to the ligand molecule 35 at the 5' end of the target molecule 30, unbound amplifier construct is removed, and labelled transcripts 70 are generated and transferred to a second solid phase vessel (not shown) for detection of the label.

In a second preferred embodiment, the signal amplification probe is an amplifier construct 5 and multivalent bridging molecule 40 in combination with a linker probe 15 for attaching the multivalent bridging molecule 40 to the target molecule 30, as shown in Figure 2. Preferably, the target molecule 30 is a nucleic acid molecule immobilized on a solid phase bead 25, coated with a receptor molecule 26. Most preferably the bead 25 is a magnetic bead, such as a Dynal M 280 SA-bead (Dynal, Great Neck, NY) . Preferably, the nucleic acid target molecule 30 has been amplified using a nucleic acid amplification system, such as PCR, in the presence of a ligand molecule 35 that becomes incorporated at the 5' end of the target molecule 30. The incorporated ligand molecule 35 binds to the receptor molecule 26 on the surface of the bead 25 to immobilize the target molecule on the bead. It will be understood by those skilled in the art that the ligand 35 can be attached to the target molecule 30 by other methods, such as covalent or chemical linking, known to those skilled in the art. Most preferably, the ligand molecule 35 is biotin and the receptor molecule 26 is streptavidin.

The linker probe 15 is preferably a nucleic acid molecule having at least one region complementary to a unique or highly variable portion of the target molecule 30 to ensure specificity of the signal amplification probe 5 for the target molecule 30. A ligand molecule 17 capable of binding to the multivalent bridging molecule 40 of the signal amplification probe 5 is incorporated at the 5' end of the linker probe 15. The ligand 17 can be the same or a different ligand than the ligand 35 incorporated at the 5' end of the target molecule 30. Most preferably, the ligand 17 is a biotin molecule. The linker probe 15 attaches to the target molecule 30, preferably by hybridization of the complementary portions of each molecule under standard hybridization conditions. In addition, the ligand molecule 17 at the 5' end of the linker probe 15 attaches to the multivalent bridging molecule 40, which is attached to the amplifier construct 5.

After removal of unbound signal amplification probe, labelled transcripts 70 are generated and transferred to a solid phase vessel (not shown) for detection of the label as described above.

In a third preferred embodiment, the signal amplification probe is an amplifier construct 5 and multivalent bridging molecule 40 used in combination with both a capture probe 10 for immobilizing the target molecule 30 to a solid phase 25 and a linker probe 15 for attaching the multivalent bridging molecule 40 to the target molecule 30, as shown in Figure 3. Preferably, the target molecule 30 is a nucleic acid molecule immobilized on a solid phase magnetic bead 25 coated with a receptor molecule 26 as described above. The advantage of this third embodiment over the first and second embodiments is that it can accommodate any target molecule 30. In other words, a ligand molecule 35, as shown in Figure 1, need not be incorporated into the target molecule 30 during amplification in order to achieve attachment to the multivalent bridging molecule 40. Therefore, less sample preparation is involved. Most preferably the target molecule 30 is amplified by PCR to achieve optimal sensitivity.

Either the capture probe 10 or the linker probe 15 is specific for a unique or highly variable portion of the target molecule 30. The remaining probe is specific for a conserved region of the target molecule 30. Preferably, the capture probe 10 is specific for a DNA sequence that is unique to or highly variable in the target molecule 30, such as a sequence specific for a particular person or ethnic group. Preferably, the linker probe 15 contains a sequence that will hybridize to a conserved region of the DNA target molecule 30. In addition, the capture probe 10 includes, preferably at the 5' end, a ligand 12 molecule capable of binding to the receptor molecule 26 with which the beads 25 are coated. Similarly, the linker probe 15 includes, preferably at its 5' end, a ligand molecule 17 capable of binding to the multivalent bridging molecule 40. If the target-reactive moiety of the linker probe or capture probe 10 is a nucleic acid moiety and the target molecule 30 is a nucleic acid molecule, hybridization of the probe moiety to the target molecule can occur if the base sequence of one is substantially complementary to the base sequence of the other. Under extremely stringent hybridization conditions, however, only oligomers that are completely complementary to each other will remain hybridized to each other. A single- stranded DNA ("ssDNA") target may be hybridized to the probe under various temperatures and conditions, according to the temperature of disassociation (Td) of the probe and the stringency required for specific binding. Hybridization techniques are well-documented in the scientific literature; one set of possible conditions is exemplified below.

Generally, the target molecule will be found in, or have been purified from, either a cell (a eukaryotic cell, a prokaryotic cell, or a plant cell) or a virus. Therefore, depending on the conditions used, complementarity between either the capture probe or the linker probe and the target molecule must extend over a region of at least 15 to 30 nucleotides in order for a stable hybrid to form. Therefore, as shown in Figure 3, the ligand 12 at the 5' end of the capture probe 10 binds to the receptor molecule 26 coating the surface of the solid phase bead 25, the capture probe 10 hybridizes to one portion of the target molecule 30 while the linker probe 15 hybridizes to a second portion of the target molecule 30, the ligand 17 at the 5' end of the linker probe 15 binds to the multivalent bridging molecule 40, the amplifier construct 5 binds to the multivalent bridging molecule 40, and labelled transcripts 70 are generated and transferred to a solid phase vessel (not shown) for detection of the label as described above. Methods of Use

As described above, the signal amplification probe is useful for the detection and quantification of target molecule in a biological sample. An assay for a target molecule, utilizing the signal amplification probe contains the following steps.

First, the sample is immobilized on a solid phase by placing the sample in proximity to a solid phase surface that has been modified to contain a receptor or capture molecule to which the target molecule will bind, either directly or indirectly. It will be understood by those skilled in the art that the target molecule can be amplified either before or after immobilization to the solid phase. For direct attachment of the target molecule, the target molecule is amplified in the presence of a ligand to incorporate a ligand at the 5' end of the target molecule and the ligand binds to a receptor or substrate with which the solid phase has been coated. For indirect attachment, a capture probe having the ligand is first bound to the solid phase and the target molecule is then bound to the capture probe, preferably by hybridization. Second, the bridging molecule and amplifier construct are combined with the immobilized target molecule and attached either to the target molecule directly, as in the first embodiment described above, or indirectly through a linker probe, as in the second and third embodiments described above. At this time, any unbound target molecule, multivalent bridging molecule or amplifier construct can be removed by washing in accordance with methods known to those skilled in the art. Third, a polymerase is added, which binds to the promoter, and the transcribable portion of the amplifier construct is transcribed. Indicator molecules, such as biotinylated nucleotide triphosphates, are preferably added simultaneously with the addition of the polymerase for incorporation of the indicator molecules into the transcripts. Finally, the transcripts are then detected in accordance with methods known to those skilled in the art. Preferably, the transcripts are harvested and transferred to a solid phase vessel coated with a receptor that binds to either the indicator molecule or the transcript and the immobilized transcripts are detected by known methods, preferably colorimetrically.

It is preferred that the target molecule be bound to the solid phase, or to the capture probe that is bound to the solid phase, before the target molecule is bound to the bridging molecule and amplifier construct. One advantage of such a procedure is that unbound bridging molecules and amplifier constructs can be washed away from bound bridging molecules and amplifier constructs to reduce background interference. Applications for Use

The signal amplification probe, and the processes and kits that employ it, are useful in nucleic acid hybridization assays, antibody-antigen assays, and other assays. Such assays are useful in the diagnosis of human disease where they can be used to detect the nucleic acids of pathogenic microorganisms and viruses, to detect the proteins that belong to or are induced by pathogenic microorganisms and viruses, to detect cancer- associated and other disease-associated molecules, and to analyze genetic material for the presence of specific genes or specific alleles of a gene.

For example, in an assay for the detection of a specific gene having a highly conserved sequence that does not vary significantly from individual to individual and a highly variable sequence that varies from individual to individual, such as an HLA allele, the following "sandwich assay" is preferred. First, a series of capture probes are immobilized to solid phase in series of microtiter plate wells or test tubes. Each set of capture probes are complementary to a different variable sequence. All the capture probes in a given well are the same. Preferably, the solid phase is a magnetic bead coated with a receptor for a ligand, and the capture probe contains the ligand and the sequence complementary to a highly variable sequence of the target molecule. Then, a sample of an individual's DNA, containing the target DNA molecule, is added to each well, so that hybridization will occur only in the well where the capture probe is complementary (in base sequence) to the particular "variable" sequence of that individual. Unhybridized molecules are washed out of the well so that, ideally, all wells except one are devoid of target molecules. Subsequently, a ligand-containing linker probe complementary to the highly conserved sequence is added to each well. It will only find target molecules in one well. As a result, when multivalent bridging molecules and amplifier constructs are subsequently added, they will only be able to find probes in one well. The amplifier constructs will bind to those probes by virtue of the fact that the probes contain ligand that will bind to the binding site of the multivalent bridging molecule (e.g., biotin). The amplifier constructs are then used as templates to generate detectable RNA transcripts as described above.

The above-described methods specify a two-step transcription process, namely binding an RNA polymerase molecule to the promoter of the amplifier construct, and transcribing part of the amplifier construct into RNA transcripts. Preferably, however, a plurality (as to number, not as to type) of RNA polymerase molecules are bound to a given amplifier construct promoter (although it is not necessary that they all be bound at the same time) and the transcribable portion of the amplifier construct is transcribed more than once so as to create multiple RNA transcripts from each amplifier construct. Signal Amplification Probe Kit

A kit containing a signal amplification probe for use in an assay for a target molecule in a sample includes the following components:

(1) a solid phase for immobilization of the target molecule;

(2) a signal amplification probe containing a multivalent bridging molecule and an amplifier construct;

( 3 ) a polymerase ; and (4) an indicator molecule for incorporation into transcripts generated by the polymerase.

The multivalent bridging molecule and the amplifier construct can be provided as a single entity as the signal amplification probe or each component can be provided separately and combined during the assay or immediately prior to use of the kit to conduct an assay. Preferably, the solid phase is pre-coated with a receptor to which the target molecule will bind, either directly or indirectly.

The kit additionally contains either:

(5) a capture probe for immobilization of the target molecule to the solid phase; or (6) a ligand and the appropriate amplification reagents for incorporation of the ligand into the target molecule.

The ligand is preferably a labeled nucleoside triphosphate (such as a biotinylated nucleoside triphosphate) for incorporated into the target molecule by a polymerase. The target molecule will then bind to the solid phase by interactions between the ligand and a receptor with which the solid phase is coated.A suitable polymerase is one that will utilize a polymerase for the nick translation process (e.g., a DNA polymerase, such as DNA polymerase I, and a deoxyribonuclease ("DNase") , such as pancreatic DNase) . Most preferably, a DNA polymerase is included in the kit for incorporation of ligand into the target molecule and an RNA polymerase is included for transcription of the amplifier construct. Furthermore, the kit can contain: (7) a linker probe for attachment of the target molecule to the multivalent bridging molecule. Either the capture probe or the linker probe, or both, can comprise a group of probes, each reactive with a different part (e.g., different nucleotide sequence) of the target molecule but each having the same ligand so that one kit can be used to detect numerous target molecules of interest with only one signal amplification probe.

In addition, the kit preferably provides a plastic microtiter plate well in which at least one well (but more likely a plurality of wells) has, covalently linked to its surface, nucleic acid molecules capable of hybridizing to RNA molecules transcribed by an RNA polymerase molecule using the amplifier construct as the template.

The signal amplification probe, assays using the probe, and kits containing the probe will be further understood with reference to the following non-limiting examples.

Example 1

Detection of Target DNA Molecules Immobilized to

Microtiter Plates Using Amplification by Signal

Amplification Probe.

As shown in Figure 1, synthetic oligonucleotide molecules, or capture probes, (10) were immobilized onto a NUNC (Roskilde, Denmark) 96-well microtiter plate (20) via a covalent linking process. Each capture probe was a DNA molecule (referred to as the 040A probe) having the following DNA sequence, wherein the 5' end of the probe is phosphorylated: 5^,-TAC TTC TAT CAC CAA GAG G-3' (Sequence Listing ID No. 1)

After capturing target DNA molecules that were labeled by biotin molecules at 5' end (30) , streptavidin molecules (40) were attached to the biotin molecules. Each target DNA molecule was a 280 bp fragment of a human cell line DNA referred to as sample 9020. The target DNA was typed as HLA DR 4 and could be recognized and bound by the 04OA capture probe described above. The target DNA was amplified by the PCR process. Biotin molecules were incorporated by using biotinylated primers during amplification. These target molecules were mixed with a known amount of irrelevant short synthetic DNA molecules (60 bases) to test the selectivity and sensitivity of the systems.

Biotinylated amplifier construct molecules (5) were immobilized to the streptavidin molecules. Each amplifier construct molecule was a partially double-stranded DNA molecule having the following

DNA sequence (Sequence Listing ID Nos. 2 and 3) :

5'-TGA CTA ATT TAA TAC GAC TCA CTA TAG-3'

3'-T TAA ATT ATG CTG AGT GAT ATC CCT CTG GAG- AAC CAC TAT CTT CAT GGA GAA CCA CTA TCT TCA-

TTT TTT TTT TT-*5'

The top strand is the plus strand (+ strand) and was biotinylated, whereas the bottom strand is the minus strand (- strand) .

After removal of any unbound amplifier construct molecules, transcription, controlled by T7 RNA polymerase (60) , was initiated. The transcripts (70) were then harvested and measured through a microtiter plate based detection system (not shown) . Immobilization of Target Molecule

Target molecules were captured by the specific capture probe in the presence of 5x SSC (sodium chloride/sodium citrate) solution by the following procedure.

1. Washed the solid phase strips of the microtiter plates (NUNC, Roskilde, Denmark) , to which the capture probes have been attached, with lOx SSC (sodium chloride/sodium citrate) three times at room temperature.

2. Added 200 μl of prehybridization solution to each well, then incubated the strips at 42°C on a heat-block overnight. The prehybridization solution contains Denhardt's reagent, SSC, SDS and salmon sperm DNA and was purchased from BioWhittaker, Inc. (Walkersville, MD) .

3. Washed the strips with lOx SSC three times at room temperature.

4. Added 30 μl of lOx SSC, 25μl denatured DNA by boiling for 15 minutes and chilled on ice, 45 μl H₂0 to each well.

5. Hybridized at 42'C on heat-block for one hour.

6. Washed three times with TNET solution at 55'C. The TNET solution contains: 50 mM Tris- HC1;25 mM NaCl; 1 mM EDTA; and 0.3% Tween; pH 7.5. Attachment of Signal Amplification Probe The signal amplification probe was attached to the streptavidin molecule via a biotin/streptavidin interaction in the presence of lx Binding & Washing buffer (B&W buffer, 10 mM Tris-HCl , 1.0 mM EDTA, and 2.0 M NaCl) as follows. 1. Added 200 μl of prehybridization solution and incubated at 42°C for 30 minutes.

2. Washed three times with TNET solution at room temperature.

3. Added 100 μl of streptavidin (SA, 1 mg/ml stock) 1:4,000 dilution diluted in lx Binding and

Washing (B&W) buffer, then incubate at room temperature for 30 minutes. A 2x solution of the B&W buffer contains: 10 mM Tris-HCl (pH 7.5); 1.0 mM EDTA; and 2.0 M NaCl. 4. Washed five times with TNET solution at 55'C. Transcription of Amplifier Construct

The amplifier construct was transcribed according to the following protocol:

1. Added 200 μl of prehybridization solution to each well, and incubate for 30 minutes at 42°C.

2. Washed three times with TNET solution at room temperature.

3. Added 1 μg of amplifier construct diluted in 24 μl H₂0 and 25 μl of 2x B&W buffer, into each well.

4. Incubated at room temperature for 30 minutes.

5. Washed five times with TNET solution at 55'C. 6. Added 50 μl of transcription mixture which consists of: lOx transcription buffer 5 μl/well lOx rNTP 2.5 μl/well

(final cone. = 0.5x) T7 RNA polymerase 1.6 μl/well

Adjust volume to 50 μl with RNase-free H₂0. The lOx transcription buffer contains: 40 mM Tris- HCl, pH 8.1; 10 mM Spermidine; 0.1% Triton X-100; 50 mM DTT; 5 mg/ml BSA; 150 mM MgCl₂; and H₂0. The rNTPs (10 mM ATP, GTP, CTP and UTP) were obtained from GIBCO-BRL, (Gaithersburg, MD) and include Biotin-¹⁴CTP.

7. Incubated at 37°C for one hour.

8. Added 1 μl of EDTA (0.5 M, pH 8.4) to each well to stop reaction.

9. Supernatant was harvested and ready for detection or stored at -20°C until further use. Detection of RNA Transcripts

Transcripts were detected by the following procedure:

1. Washed strips, to which probes specific for the transcripts have been attached, three times with lOx SSC at room temperature.

2. Added 200 μl of lx uniblock reagent (Analytical Genetic Testing Center, Inc. , Denver, CO) to each well, and incubated at room temperature for one hour.

3. Washed three times with lOx SSC at room temperature.

4. Added 30 μl of lOx SSC, 25 μl of diluted RNA (2 to 10 μl RNA transcripts per well, diluted in H₂0) , and 45 μl of H₂0 to each well.

5. Incubated at 42'C for one hour.

6. Washed three times with TNET solution at 55°C.

7. Added 100 μl of streptavidin-alkaline phosphatase (SA-AP) in lx uniblock to each well.

8. Incubated at room temperature for 30 minutes.

9. Washed three times with TNET at room temperature. 10. Added 100 μl of the chromogen PNPP (p- nitrophenyl phosphate) , diluted in DEA solution, to each well (one 5 mg tablet/5 ml DEA solution) . The DEA solution contains: 10 mM diethanolamine; and 0.5 mM MgCl, pH 9.5. 11. Read on ELISA microtiter plate reader.

Determination of Specificity in Microtiter Plate Based Format:

Figure 1 clearly demonstrates that two streptavidin/biotin interaction events occur in this system. The first interaction is between biotinylated target DNA molecules and streptavidin (Specificity I) ; and the second interaction is between streptavidin and biotinylated amplifier construct molecules (Specificity II) .

To determine Specificity II, the efficiency of signal amplification of biotinylated amplifier construct molecules was compared with the efficiency of non-biotinylated amplifier construct molecules, which are known to be functional in a tube reaction format. This was done to ensure that the non-biotinylated amplifier construct is functional.

Figure 4 shows that, while successful transcription is detected with biotinylated amplifier construct molecules, the transcription efficiency is reduced significantly to only background level with non-biotinylated amplifier construct molecules.

To determine Specificity I, the following inhibition method was used. Streptavidin-alkaline phosphatase was used instead of streptavidin molecules in the assay. Since alkaline phosphatase is about twice as big as streptavidin, the binding of biotinylated amplifier construct must be inhibited to certain extent. Figure 4 clearly shows that when streptavidin-alkaline phosphatase is used, the signal amplification efficiency is reduced to only background level.

Determination of Specificity and Selectivity In Microtiter Plate Based Format. To determine the selectivity and sensitivity of the signal amplification probe in microtiter plate format, various amounts of target DNA molecules were mixed with 10¹³ irrelevant synthetic DNA molecules (fragment size=60 bases) , and detected by both the signal amplification probe and regular plate based hybridization method. While regular plate based hybridization method only detected 2.5 x 10⁹ molecules (data not shown), the signal amplification probe was capable of detecting 2.5 x 10⁶ molecules as shown in Figure 5. Note that only 10 μl of a total 50 μl volume of transcripts were used for detection in each sample. Thus, a minimum of 1,000 fold increase of sensitivity was established using the signal amplification probe in comparison with regular plate based hybridization method. Under optimal conditions, a detection of 2.5 x 10⁵ molecules was observed using the signal amplification probe as shown in Figure 6.

Therefore, approximately 10^s to 10⁶ target DNA molecules are detectable by the signal amplification probe in this experimental system. Conclusion of sensitivity determination In the microtiter plate based format, a minimum of 1,000 fold increase of sensitivity was observed with the signal amplification probe shown in Figure 1 in comparison with a regular microtiter plate-based hybridization method. The detection range of 10⁵-10⁶ target DNA molecules (approximately 0.02 -0.16 pg DNA) was established with the signal amplification probe, while only 10⁹ molecules were detectable using regular plate based hybridization method.

Example 2 Detection of Target DNA Molecules Immobilized on Streptavidin-coated Beads Using Amplification by Signal Amplification Probe.

As shown in Figure 2, target DNA molecules (30) which are labeled by biotin molecules (35) at the 5' end are attached to magnetic streptavidin- coated beads (25). (Dynal, Inc., Great Neck, NY) Biotinylated sequence specific DNA probes (15) are then hybridized to the target DNA molecules. Streptavidin molecules (40) are attached to hybridized probes via biotin/streptavidin binding. Biotinylated amplifier construct molecules (5) are then immobilized to the streptavidin molecules.

After removal of any unbound amplifier construct molecules, the transcription controlled by T7 RNA polymerase (60) is initiated. The transcripts (70) are then harvested and measured through a microtiter plate based detection system (not shown) . Immobilization of Target Molecule to Beads 1. Twenty μl of M-280 streptavidin (SA) beads (Dynal Inc., Great Neck, NY) were added into a siliconized 1.7 ml microcentrifuge tube (Costar Corporation, Cambridge, MA) . Sixty μl of 2X B&W buffer were added into the tube. The beads were resuspended and washed by pipetting up and down for several times. A DYNAL MPC-E™ apparatus (Dynal Inc.) was then used to separate the beads. Additional four times washing was conducted with 80 μl of 2x B&W buffer each time. 2. Washed beads were resuspended in 40 μl of 2x B&W buffer, and heat-denatured biotinylated target DNA molecules (denatured by boiling for 10 minutes, and chilled on ice) were added into each tube. The target DNA molecules were the same (sample 9020) as described above in Example 1.

3. The beads were resuspended and incubated at room temperature for 15 minutes on a shaker with a low speed motion.

4. The beads were washed 5X as described in Enzymes. 3rd Edition, Paul D. Boyer Editor. Vol.

15, Nucleic Acids, Part B, Chapter 4, 1982, and resuspended in 40 μl of 2X B&W buffer.

5. Twenty μg of biotinylated DECT-B oligonucleotide molecules (Synthesized by Oligo etc. Inc., Wilsonville, OR) in 40 μl of H₂0 were added into each tube to saturate any residual streptavidin sites on the beads after binding of the target molecule. The sequence of the DECT-B molecules is as follows:

5'-Biotin-CCT AAT CGG TAA GCC TAC GTT CGA GTT- GAC CAA GCT GAT CTT AGG CAT ACC GTG CAC TGT-3' (Sequence Listing ID No. 4)

6. The beads were resuspended and incubated at room temperature for 15 minutes on a shaker with a low speed motion.

7. The beads were washed 5X, as described above in step 4, and resuspended in 30 μl of lOx

SSC. Forty μl of biotinylated linker DNA probes (0.1 μg/tube in H₂0) were added into each tube. An additional 20 μl of H₂0 was added into each tube to make the final volume to 100 μl. 8. The beads were resuspended and incubated at 42°C on a heat block for 1 hour.

9. The beads were washed 5X as described in step 4 above and resuspended in 40 μl of IX B&W buffer. 10. Forty μl of streptavidin (1:40,000 of 1 mg/ml) in IX B&W buffer was added into each tube. The beads were resuspended and incubated at room temperature for 15 minutes on a shaker with a low speed motion. 11. The beads were washed 5X as described in step 4 above and resuspended in 40 μl of 2x B&W buffer. Forty μl of short, biotinylated amplifier construct (AC) (0.2 μg/tube in H₂0) were added into each tube. The amplifier construct molecule was the same as described above in Example 1.

12. The beads were resuspended and incubated at room temperature for 15 minutes on a shaker with a low speed motion.

13. The beads were washed 5X as described in step 4 above and added with 50 μl of transcription reaction mixture (described above in Example 1) . 14. The beads were resuspended and incubated in a 37°C water-bath for 30 minutes.

15. The supernatant containing RNA transcripts was isolated and used for microtiter plate based detection (5 μl/well was used for detection). The supernatant was stored at -20'C if the detection was not performed on the same day.

Detection was performed essentially as described above in Example 1. Determination of Specificity in Dvnal M 280 SA- beads Based Format:

Two levels of specificity are also defined in this format. Specificity I defines the interaction between streptavidin and biotinylated DNA targets. Specificity II defines the interaction between streptavidin and biotinylated amplifier construct DNA molecules.

To determine the specificity I, two approaches were taken. First, when biotinylated irrelevant oligonucleotides were used to block the binding of target molecules to streptavidin beads, signal amplification efficiency was reduced to only 20% of non-blocked samples, as shown in Figure 7. Second, non-biotinylated amplifier construct molecules were also used as a control, and signals were barely detected, also shown in Figure 7. Determination of Specificity and Selectivity in

Dynal M 280 Streptavidin-Beads Based Format:

The target DNA molecules, described above in Example 1, were used to detect the selectivity and sensitivity of the signal amplification probe in a streptavidin beads model. Approximately 2 x 10⁶ target DNA molecules were clearly detectable using the signal amplification probe as shown in Figure 8, which indicates a 1,000 fold increase of sensitivity in comparison to regular plate based hybridization method. Note that only 5 μl of a total volume of 50 μl of RNA transcripts were used for detection in each sample.

Conclusion of Sensitivity Determination

In the streptavidin-beads based format, the detection sensitivity was approximately at 10⁶ target DNA molecules (approximately 0.08 pg. DNA).

Quantitative Results Obtained Using the Signal Amplification Probe

Figures 5, 6, 7, and 8 clearly demonstrate the potential of constructing a standard curve to facilitate the quantitation of target DNA molecules in any sample.

In addition, contamination of a few molecules in a negative sample is unlikely to cause false positive. It is also feasible to construct a standard curve to quantitate the target molecules in a sample population. Therefore, as long as the sensitivity level is acceptable for certain tests, quantitation should be feasible.

Modifications and variations of the signal amplification probe and methods of use will be obvious to those skilled in the art from the foregoing description. Such modifications and variations are intended to come within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: GenTrak, Inc.

(B) STREET: 5100 Campus Drive

(C) CITY: Plymouth Meeting

(D) STATE: Pennsylvania

(E) COUNTRY: U.S.

(F) POSTAL CODE (ZIP) : 19462

(G) TELEPHONE: (215)825-5115 (H) TELEFAX: (215)941-9498 (I) TELEX: 255935

(ii) TITLE OF INVENTION: Signal Amplification

Probe and Methods of Use

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/893097

(B) FILING DATE: 29-MAY-1992

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Synthetic

(ix) FEATURE:

(A) NAME/KEY: misc_binding

(B) LOCATION: 1..19

(D) OTHER INFORMATION: /note= "Probe for HLA DR 4" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TACTTCTATC ACCAAGAGG

19

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Synthetic

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..5

(D) OTHER INFORMATION: /note= "Leader Sequence"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 6..27

(D) OTHER INFORMATION: /note= "RNA Promoter + Strand"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

TGACTAATTT AATACGACTC ACTATAG

27

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 75 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE:

(A) ORGANISM: Synthetic

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 54..75

(D) OTHER INFORMATION: /note= "RNA Promoter - strand hybridized to Seq. No. 2 nucleotides 6-27"

(ix) FEATURE:

(A) NAME/KEY: misc_feature

(B) LOCATION: 1..53

(D) OTHER INFORMATION: /note= "Transcription Template"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TTTTTTTTTT TACTTCTATC ACCAAGAGGT ACTTCTATCA CCAAGAGGTC TCCCTATAGT 60

GAGTCGTATT AAATT

75

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 60 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Synthetic Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CCTAATCGGT AAGCCTACGT TCGAGTTGAC CAAGCTGATC TTAGGCATAC CGTGCACTGT 60

Claims

We Claim:

1. A signal amplification probe comprising: a) a transcription-based amplifier construct, and b) a multivalent bridging molecule for attachment of the amplifier construct to an immobilized target molecule, wherein the amplifier construct includes a ligand that binds to the multivalent bridging molecule and the target molecule is immobilized on a solid phase.

2. The signal amplification probe of claim 1 wherein the amplifier construct is a double- stranded nucleic acid molecule comprising an RNA polymerase promoter and a template for transcription by an RNA polymerase binding to the promoter.

3. The signal amplification probe of claim 1 wherein the double-stranded nucleic acid molecule is DNA.

4. The signal amplification probe of claim 1 wherein the multivalent bridging molecule is selected from the group consisting of a chelating agent, avidin, streptavidin, digoxigenin and a carbohydrate.

5. The signal amplification probe of claim 1 wherein the multivalent bridging molecule is streptavidin.

6. The signal amplification probe of claim 1 wherein the ligand is biotin.

7. The signal amplification probe of claim 1 further comprising a linker probe for attachment of the multivalent bridging molecule to the target molecule, wherein the linker probe includes a ligand that binds to the multivalent bridging molecule and a binding moiety that binds to the target molecule.

8. The signal amplification probe of claim 7 wherein the target molecule is a nucleic acid molecules and the binding moiety of the linker probe is a nucleic acid molecule having a sequence complementary to a sequence of the target molecule.

9. The signal amplification probe of claim 7 wherein the ligand of the linker probe is biotin.

10. The signal amplification probe of claim 1 wherein the solid phase is selected from the group consisting of the surface of a solid phase vessel or a solid phase bead.

11. The signal amplification probe of claim 1 wherein the solid phase is coated with a receptor.

12. The signal amplification probe of claim

11 further comprising a capture probe for immobilization of the target molecule to the solid phase, wherein the capture probe includes a ligand that binds to the receptor with which the solid phase is coated and a binding moiety that binds to the target molecule.

13. The signal amplification probe of claim

12 wherein the target molecule is a nucleic acid molecule and the binding moiety of the capture probe is a nucleic acid molecule having a sequence complementary to a sequence of the target molecule.

14. A method for detecting a target molecule in a sample comprising the steps of: a) immobilizing the target molecule to a solid phase; b) adding a signal amplification probe to the immobilized target molecule, wherein the signal amplification probe comprises a transcription-based amplifier construct and a multivalent bridging molecule that binds to both the target molecule and the amplifier construct; c) removing unbound signal amplification probe; d) transcribing the amplifier construct with a polymerase to produce transcripts; and e) detecting the transcripts.

15. The method of claim 14 wherein the transcripts are transferred to a solid phase vessel coated with a receptor specific for the transcripts prior to the detection step.

16. The method of claim 14 wherein the solid phase is coated with a receptor.

17. The method of claim 16 wherein the target molecule is first amplified and a ligand specific for the receptor is incorporated into the target molecule during amplification.

18. The method of claim 16 wherein the target molecule is immobilized on the solid phase by a capture molecule having a binding moiety that binds to the target molecule and a ligand that binds to the receptor with which the solid phase is coated.

19. The method of claim 14 wherein the multivalent bridging molecule binds to the target molecule by a linker molecule having a binding moiety that binds to the target molecule and a ligand that binds to the multivalent bridging molecule.

20. The method of claim 18 wherein the multivalent bridging molecule binds to the target molecule by a linker molecule having a binding moiety that binds to the target molecule and a ligand that binds to the multivalent bridging molecule.

21. The method of claim 20 wherein the target molecule is an HLA gene, the capture probe is a nucleic acid molecule having a sequence that hybridizes to a non-conserved sequence of the HLA gene, and the linker probe is a nucleic acid molecule having a sequence that hybridizes to a conserved sequence of the HLA gene.

22. A signal amplification probe kit comprising: a) a solid phase for immobilization of the target molecule; b) a signal amplification probe containing a multivalent bridging molecule and an amplifier construct;

3) a polymerase; and

4) an indicator molecule for incorporation into transcripts generated by the polymerase.

23. The kit of claim 22 further comprising a capture probe for immobilization of the target molecule to the solid phase.

24. The kit of claim 22 further comprising a ligand and the appropriate amplification reagents for incorporation of the ligand into the target molecule.

25. The kit of claim 22 further comprising a linker probe for attachment of the target molecule to the multivalent bridging molecule.

26. The kit of claim 22 further comprising a second solid phase for capture of transcripts generated by the polymerase.