WO2003064692A1

WO2003064692A1 - Detecting and quantifying many target nucleic acids within a single sample

Info

Publication number: WO2003064692A1
Application number: PCT/US2002/002372
Authority: WO
Inventors: Qiang Wan
Original assignee: Atlantic Biolabs, Inc.; Chengdu Advancetech Biotechnologies Co., Ltd
Priority date: 2002-01-29
Filing date: 2002-01-29
Publication date: 2003-08-07
Also published as: CN1617936A

Abstract

In a single reaction, thousands of different nucleic acid sequences can be detected and quantified, with a sensitivity to as few as 10 target copies per sample, by a method that incorporates specific hybridization and rolling circle amplification. This approach offers greater accuracy because the amount of reaction product generated at the end of amplification is strictly proportional to and dependent on the amount of target nucleic acid, and sample-to-sample variability is eliminated through the use of a universal template and primer. The approach can be adapted to the detection of point mutations as such and to identifying the presence of genetic mutations for purposes of research and diagnosis, respectively.

Description

DETECTING AND QUANTIFYING MANY TARGET NUCLEIC ACIDS WITHIN A SINGLE SAMPLE

FIELD OF INVENTION

The present invention relates to the field of genetic analysis and, in particular, concerns using hybridization and rolling-circle amplification techniques for the accurate detection and sensitive quantification of nucleic acids, including mutant nucleic acids, within a sample. The compositions and methodology of the present invention are particularly useful in detecting the presence and the copy number of multiple nucleic acids within a sample, for research purposes, personal identification, and diagnosis, among other purposes.

BACKGROUND OF THE INVENTION

The accurate detection and quantification of nucleic acids within a sample is a valuable tool for a variety of applications, including research, medical diagnosis, new drug discovery, and other aspects of the life sciences and biomedicine. Nucleic acid detection also presents many practical aspects including personal identification for such purposes as paternity testing, forensic science, organ-transplant donor-recipient matching and pre-natal counseling.

Genetic mutations account for a vast number of diseases including numerous forms of cancer, vascular disease, neuronal and endocrine diseases. Many such genetic mutations have been identified including the variations in nucleic acid sequence that lead to the diseases of cystic fibrosis, muscular dystrophy, Tay-Sachs disease, hemophilias, phenylketonuria and sickle-cell anemia. Since genetic variations are connected to disease states, the detection of the exact nucleic acid sequence present within a sample is crucial both pre- and post- symptomatically for accurate medical diagnosis, prophylactics, and treatment.

Genetic mutations can be as small as a single base-pair mutation in a gene, but also include multiple base pair changes, insertions, deletions, and translocation. Among over 500 recognized genetic diseases, many can be traced to single base-pair mutations. The prime example of a single base pair change that results in aberrant cellular activity and disease is sickle-cell anemia. Single base-pair mutations are the hardest to detect as discriminating a single base pair difference among a large pool of target nucleotide sequences in a sample is scientifically challenging. As such, a method to detect a single base pair difference among many sequences is crucial for research and for ameliorating the effects of genetic diseases.

Other variations in the genetic code may also change the functionality of some aspect of life and lead to disease. Of these variations, changes in the gene product or in the expression levels of the genes themselves can be the determining factor. Changes in gene expression can be through changes in the copy number of the genetic DNA (e.g. gains and losses of genetic material result in malignant transformation and progression) or through changes in levels of transcription (e.g. through control of initiation, changes in the RNA precursors, aberrant RNA processing, etc.). Therefore, the quantification of the expression level or copy number of a nucleic acid can be essential to medical diagnosis.

The ability to discern between nucleic acids containing only a one base pair difference within a pool of many genes is essential to the analysis of genetic material and the quantification of such provides additional utility. As extensively and clearly presented in US Patent No. 6,268, 1 47, a variety of technical approaches and strategies have been devised for simultaneously analyzing multiple nucleic acid sequences within a single sample containing a large number of sequences. The analytical approach most commonly used is hybridization techniques that rely on nucleic acid capture and labeling.

One such hybridization technique utilizes probe nucleic acids, which are complementary to the target nucleic acid, to locate and hybridize to the target nucleic acids within the sample. Either the probe or target nucleic acid is pre-labeled with a detectable reporter moiety such as a fluorescent molecule or radioisotope. The presence of label remaining after hybridization and a washing step represents the presence of target DNA found within the sample. Dot blots and slot blots use this simple technique. A variation of this technique takes the form of membrane hybridization protocols, whereby, a mixture of DNA fragments is separated by gel electrophoresis then transferred to a membrane and immobilized. By reacting the membrane-immobilized DNA fragments with one or more labeled probes under specific hybridization conditions, the presence or absence of the nucleic acid of interest can be ascertained. Such membrane techniques include simple nucleotide polymorphism detection (SNP), and Northern and Southern hybridization.

A limitation to this approach is the need for extra steps to incorporated label into either the target probe or the detection probe. Although labeling strategies are straightforward and widely practiced, they nevertheless require additional steps, and, if a large number of samples need to be analyzed, the additional labeling steps can be time- consuming and expensive. Moreover, in order for a positive signal to be detected using nucleic acid probe labeling, the sample must contain at least a hundred thousand to a million copies of the target nucleic acid to be of sufficient intensity to be detected using standard techniques. Thus, these systems are not sensitive to low copy number nucleic acids and can not be used for targets that amount to less than a hundred thousand to a million copies within the sample. As a result, since most genes in the genome are present in the cell at copy numbers between one and a few copies, this system alone is incapable of determining the presence or absence of the great majority of nucleic acids.

Another limitation of hybridization techniques is that they can not monitor or determine gene expression level or determine copy number. These hybridization methods can only determine the presence or absence of a sequence of interest, not the quantity of the sequence. Furthermore, single-base pair differences in the sequence are not always distinguishable. This is so because only a minor difference in binding energy separates target and non-target probes containing only a one-base pair difference. Thus, the stringency of the hybridization, incubation, and washing steps must be carefully controlled in order to be sensitive enough to discriminate between the matched probe and the single nucleotide mismatched target probe. Optimal reaction conditions will therefore vary greatly depending on the target. As a result, each reaction must be specific for a particular target and probe interaction making the screening of more than one nucleic acid simultaneously in automated and/or high volume processes difficult.

In general, this approach is not sensitive enough for nucleic acid sequences found in low numbers for such applications as analyzing medical samples where only a few cells can be taken for sample analysis. Additionally, this method alone is not suitable for determining the presence of minor changes in sequences (mutations) that lead to disease among a large number of sequences. Another method for detection and quantification of nucleic acids uses the polymerase chain reaction (PCR) to ascertain the presence of a target nucleic acid within the sample. In the PCR approach, primers complementary to sequences 5' and 3' regions of the target sequence of interest are used to amplify the target sequence of interest via a thermal cycling reaction. The amplified nucleic acids sequences can then be determined by a variety of techniques, e.g. gel electrophoresis, to ascertain the presence or absence of a specific sequence within a sample. This approach is particularly useful for detecting low copy number of the target sequences interspersed within a sample containing a multiplicity of sequences. This is the case because more label can be incorporated through the exponential PCR enzymatic process such that positive signals are detected at low copy numbers. However, this technique has limitations in the context of determining copy number of multiple targets in a single sample, as well as in determining single-base pair mutations.

The PCR process is a highly sensitive enzymatic reaction. Variations in the annealing rates of the primers in the reaction as well as varying polymerase extension rates for each sequence due to salt concentrations and annealing temperatures will affect the amount of product generated in the reaction. This sample-to-sample variability makes this method unsuitable for determining variations in copy number of multiple targets in a sample in a single reaction.

And additional limitation to using PCR to assay for multiple nucleic acids is the requirement for a different set of PCR primers for each target sequence of interest. As each new primer set is added to the reaction, the number of potential amplicons and primer dimers which could form increases exponentially. It has been shown that more than multiple pairs of primers in a PCR reaction lead to a high rate of non-specific amplification and false positives. See Sambrook et al., MOLECULAR CLONING : A LABORATORY MANUAL (2^nd ed.), pages 1 4 and 1 5 (Cold Spring Harbor Laboratory Press, 1 989). Additionally, when both wild-type and mutant are present, PCR can result in the amplification of both, thereby necessitating a follow up step to isolate and discern between the presence of mutant form and wild-type.

As discussed in U.S. patent No. 6,31 2,892, nucleic acid detection also is performed using probe ligation methods to identify known sequences. Probe ligation methods involve two probes that span a target region of interest. The probes are complementary to and hybridized with the target such that the 3' end of the first upstream probe is immediately adjacent to the 5' end of the second downstream probe. The abutting ends of the probes can be joined by ligation, if and only if there is perfect complementarity at the junction, i.e. , no mismatch at the intersection of the abutting probes. The ligation reaction, which covalently modifies the probes from two nucleic acid strands to one nucleic acid strand, is extremely sensitive in that a single base pair mismatch at the junction point will prevent ligation of the probes. The ligated probe then can be assayed, indicating the presence or absence of the exact target sequence. See also U.S. patent No. 4,833,750.

This technique is specific, in that the ligase will not join the DNA strands when there is even a single base-pair difference between the target and the probe. Yet the probe ligation method alone cannot generate enough product to detect and quantify small amounts of target sequences.

Because, the ligation event itself does not provide sufficient product for detection, probe ligation techniques require an additional amplification step to detect the presence of a positive ligation reaction. The literature in this area illuminates numerous detection methods. Many of the current approaches rely on labeling the probe DNA with fluorescent or radioactive probes. See U.S. patent No. 4,833,750. Others use PCR amplified targets and/or multiple cycles of hybridization and ligation reactions. See, for example, U.S. patent No. 6,31 2,892. Still, conventional techniques suffer from an inability to detect or quantify small amounts of target sequences.

Detection systems for the probe ligation method that use labeled probes have the same limitation of label density and inability to detect targets in low copy numbers in the sample as found in the standard hybridization techniques detailed above. Thus, these techniques alone are not able to present a detectable signal for nucleic acids that exist in low copy numbers.

Detection techniques that use PCR and other amplification reactions, such as multiple rounds of hybridization and ligation, are limited because these amplification methods are dependent upon nucleic acids that vary depending on the target nucleic acid of interest. Because of this fact, each detection of a different target sequence will have distinctive reaction kinetics and the quantity of amplification product will vary accordingly. The variation between targets and the resulting variation in signal makes this method unsuitable for accurate quantification of multiple nucleic acids in a sample.

Additional PCR amplification methods are not well suited for the microarray format. Nucleic acid microarrays have the advantage of allowing for the analysis of hundreds and thousands of nucleic acid sequence. Each sequence to be tested is printed in micrometer dimensions to a specific region of a solid surface. The nucleic acids are not separated by physical partitions. In the PCR amplification method, however, the amplified product of the PCR reaction dissociates into solution. Thus, any amplified product from the PCR amplification will not be found localized to the same region of the solid support as where the nucleic acid to be tested was placed. As such, separated physical spaces are necessary for each reaction such that positive signals generated by PCR amplification can be connected to the request positive nucleic acid. Because microarray formats do not have physical partitions between nucleic acid sequences, the PCR amplification method can not efficiently screen hundreds and thousands of nucleic acid sequences in a single sample.

In light of the deficiencies of current techniques for analyzing nucleic acids, it is clear that there remains a need for a rapid single assay format to detect the presence or absence of multiple selected sequences in a nucleic acid sample containing a diversity of non-target sequences even when those samples are in low abundance and a method of accurate quantification for the same.

SUMMARY OF THE INVENTION

The present invention addresses the need for sensitively detecting and quantifying as many as thousands of different nucleic acid sequences in a single sample within a single reaction. The invention has greater sensitivity than hybridization methods and can detect as few as 1 0 copies of a target within a sample by a method that incorporates the probe ligation technique with rolling circle amplification.

The present invention also addresses the need for greater accuracy in detecting and quantifying as many as thousands of different nucleic acid sequences in a single reaction. Using the rolling circle technology the single positive ligation event is amplified ten thousand fold in a manner that is strictly proportional to and dependent on the copy number of target nucleic acid. Moreover, sample-to-sample variability is eliminated through the use of a universal template and primer. As a result, this method accurately and precisely determines the quantity of target within the sample even when the target is at a low copy number.

In addition, the present invention contemplates detection of point mutations within a sample. This detection can be used to identify the presence of genetic mutations for the purpose of research or diagnosing human disease. This method is both sensitive and accurate as it can detect a one base pair difference between target and non-target nucleic acid with an accuracy of at least 99.9% .

To these and other ends there is provided in one aspect of the present invention, a method for analyzing nucleic acids by way of hybridizing a target nucleic acid to a capture probe, hybridizing a counting probe to the target nucleic acid, ligating the capture probe to the counting probe, and hybridizing a single-stranded, circular DNA to counting probe, and then amplifying the circular DNA, where the conditions for amplification are sufficient for detecting the presence of target nucleic acid.

In another embodiment of the present invention, the target nucleic acid is DNA, RNA, or cDNA. In yet another embodiment of the present invention, the target nucleic acid is from a bacterium, a virus, a parasite, or a fungus.

In accordance with another aspect of the present invention, the target nucleic acid sample is taken from body fluid, tissue, or feces.

In accordance with yet another aspect of the present invention, the target nucleic acid sample is taken from blood, saliva, urine, or sputum.

In a further aspect of the present invention, the capture probe is complementary to a nucleic acid sequence from a bacterium, a virus, a parasite, or a fungus. In another aspect, the present invention can determine the quantity of target nucleic acid present by comparing the quantity of amplification product to at least one known quantity of nucleic acid standard.

In a preferred embodiment, the present invention provides a method for analyzing a target nucleic acids by hybridizing a target nucleic acid to a capture probe and hybridizing a counting probe to the target nucleic acid, such that a hybrid comprised of capture probe, counting probe, and target probe is formed and exposing the hybrid to a ligase and then washing away counting probe and nucleic acid not covalently linked to the capture probe; then adding a single-stranded, circular DNA to the capture probe and adding to the capture probe a nucleic acid polymerase that can amplify single-stranded circular DNA; and then ascertaining whether amplified circular DNA is present or absent.

Another preferred embodiment of the invention includes a molecular assemblage of a capture probe, a counting probe, and a single-stranded, circular DNA segment where the circular DNA segment is contiguous with the counting probe.

In yet another embodiment of the present invention, the target nucleic acid of the molecular assemblage is DNA, RNA, or cDNA.

In accordance with another embodiment of the present invention, the target nucleic acid of the molecular assemblage is from a bacterium, virus, parasite, or fungus.

In another embodiment of the present invention, the capture probe of the molecular assemblage is complementary to a nucleic acid sequence from a bacterium, a virus, a parasite, or a fungus. In yet another embodiment, the target nucleic acid of the molecular assemblage is from body fluid, tissue, or feces.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of a DNA chip or a 96-well plate on which capture probes are immobilized.

Figure 2 is a diagram that illustrates capture of specific targets among a mixture of targets by nucleic acid hybridization. The targets B and D are hybridized specifically with the capture probes B and D, respectively. The counting probes B and D are annealed to the targets B and D, respectively. As a result, a molecular complex is formed comprised of three nucleic acid molecules: capture probes, counting probes, and a target molecule. The counting probe has a phosphorous group at its 5'-end, which is required for a ligation reaction. The nonspecific targets and extra indicator probes remain free. Figure 3 indicates that the counting probes B and D are covalently connected with the capture probes after ligation reaction catalyzed by DNA ligases in the presence of ATP. The extra counting probes and nonspecific targets continue to remain free.

Figure 4 illustrates that the non-interest target molecules and free counting probes are removed by wash. The counting probes, which were annealed to the targets before the wash, remain on the solid surface because of the covalent linkages with the capture probes.

Figure 5 shows that a single-stranded, circular DNA molecule, such as M 1 3 ssDNA, is annealed with the 3'-end portion of the counting probes, which contains sequences complementary to a portion of the single stranded circular DNA molecule.

Figure 6 is a schematic diagram indicating a linear rolling circle amplification reaction and a large amount of labeled DNA product generated at the end of the rolling circle amplification reaction. Figure 7 illustrates the quantification of DNA products generated at the end of an amplification reaction, pursuant to the invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The inventors have devised an approach for sensitively and accurately detecting and quantifying thousands of different nucleic acid sequences in a single reaction. The invention has greater sensitivity than hybridization methods and can detect as few as 1 0 copies of a target within a sample by a method that incorporates a variation of the probe ligation method with rolling circle amplification.

The invention affords greater accuracy than conventional methodologies because the quantification of the reaction is strictly proportional to and dependent upon the copy number of target nucleic acid, and sample-to-sample variability is eliminated through the use of a universal template and primer. Moreover, the technology of nucleic acid microarrays, such as DNA chips, is advanced not only because numerous nucleic acids can be screened concomitantly but also because the nucleic acids can be detected and quantified, with a high degree of precision.

In general terms, a probe-ligation of the present invention employs two probes, which are amenable to ligation when they are adjacent one another on a corresponding target nucleic acid, to capture a target nucleic acid in a hybridization reaction. These two probes, here referred to as "a capture probe" and "a counting probe, " are ligated in the presence of complementary target nucleic acid, thereby to form a single probe, which represents the presence of one target.

The 3' end of the counting probe, according to the present invention, is unique in that it contains a sequence that is complementary to a sequence of a single-stranded circular DNA, such as M 1 3, such that the counting probe can act as a primer for a rolling-circle amplification reaction of single-stranded DNA that is hybridized to the counting probe. Thus, the present invention applies linear rolling circle amplification, in contrast to the approach taken in conventional ligation techniques, such as amplifying a probe or target, which varies with every target DNA, or performing multiple rounds of probe-to-target hybridization and ligation.

In the presence of circular DNA, a nucleic acid polymerase, and nucleotides, an amplification results in a long nucleic acid molecule that contains hundreds of copies of the circular DNA sequence, which, in accordance with the present invention, remain attached to the immobilized probe DNA. By means of a single primer, rolling circle amplification generates hundreds of tandemly linked copies of the circular template within a few minutes. Up to 1 0⁴ copies of the original single- stranded, circular DNA, covalently linked to the immobilized probe DNA, can be produced at the site of a single ligation event, i.e. , the site where a single target molecule is detected. Zhong et al. , Proc. Nat'l Acad. Sci. USA 98: 3940-945 (2001 ).

More specifically, the counting probe includes a 5' end, which contains a region complementary to the 3' end of the target DNA, and a 3' end that contains a region complementary to the sequence of a single- stranded, circular DNA. The circular DNA can be any nucleic acid sequence, other than sequences homologous to the capture probe, of which M 1 3 is illustrative, and can be generated by any one of several well-known means. The 3' end of the counting probe, once hybridized to the single-stranded, circular DNA, can serve as a primer for the amplification reaction. In this context, polymerase and nucleotides can be added to generate an extended nucleic acid molecule that is primed from and, hence, covalently attached to the 3' end of the counting probe. The resultant amplified product is covalently attached to the solid support through the covalent attachment to the probe. This product of the rolling-circle amplification can be detected and quantified in a variety of ways, e.g. , through the use of radionucleotides, fluorescence-labeled nucleotides, or other labeling strategies. In any event, the present invention exploits rolling-circle amplification via a signal-detection strategy that replicates circularized nucleic acids with either linear or geometric kinetics, under isothermal condition. See Lizardi et al. , Nature Genet. 1 9: 225-32 (1 998).

By virtue of its rolling-circle aspect, the present invention benefits from a signal amplification that does not vary with each sample, in contrast to PCR-based techniques and methods that employ multiple rounds of hybridization and ligation. The primer and the single stranded circular DNA to be amplified are identical in each reaction, for every target. Accordingly, each probe has an equal chance and equivalent reaction kinetics for annealing to the single stranded circular DNA and priming the rolling circle detection reaction. As a result, there is considerably less variability in the detection step; each probe will be amplified equally, ensuring accurate quantification and consistent results, irrespective of the target sequence.

In accordance with the present invention, moreover, thousands of reactions can be carried out in the same multi-well plate or on a DNA chip or other microarray, because the amplified product is attached covalently, through the probe, to the solid support. This allows for the localization of the signal-amplification product to the same region of the support where the capture probe was contained originally. (In PCR techniques, by contrast, the amplified product dissociate into solution.) With its ability to localize precisely a signal arising from a single ligation reaction, the method of the present invention is well-suited for microarray formats. Furthermore, the rapid and substantial generation of DNA allows for simple, accurate signal-amplification detection, capable of identifying a nucleic acid that is present in as few as 1 0 copies per sample.

This detection method has been applied to a modified version of a well-known probe ligation method for detecting the presence of target nucleotide. See U.S. patents No. 6,31 2,892 and No. 4,883,750, and also Wu et al. , Genomics 4: 560 (1 989), Landegren et al. , Science 241 : 1 077 (1 988), and Winn-Deen et al. , Clin. Chem. 37: 1 522 (1 991 ). In general, in the presence of an exact nucleic acid match within the sample, the probe ligation method creates a detectable immobilized probe. The method of the present invention modifies other probe ligation detection methods in that the 3' end of the counting probe contains sequences commentary to sequences in a single stranded circular DNA molecule. A covalently ligated probe containing both the 5' immobilized end and the 3' single stranded circular DNA complementary end is only generated in the presence of target DNA in the sample. Additionally, because of the specificity of the ligation enzyme, the presence of a mismatch at the junction will prevent ligation when hybridized to any other nucleic acid in the sample including those with only a one basepair difference.

Specifically, in this method, a first nucleic acid probe, the capture probe, is immobilized to a solid support. The sequence of the capture probe is designed to be complementary to a conserved region of the target sequence. The 5' end of the capture probe is constructed so that it has an active chemical group, such as biotin or a primary amine with a linker, that will facilitate immobilization of the capture probe to a surface. The capture primer is then immobilized to a support made of glass, plastic, or any other solid surface through the 5' end of the capture probe. A second nucleic probe, the counting probe, is designed so that the 5' portion of the probe is complementary to a region on the target sequence located immediately upstream to the region of the target where the capture probe will anneal. The 3' end of the counting probe is unique in that it is designed to contain a sequence that is complementary to a sequences of a single stranded circle DNA (such as M 1 3) so that the counting probe can act as a primer for a rolling circle amplification reaction of single stranded DNA hybridized to the counting probe. The counting probe also contains a 5' phosphate group.

The target nucleic acid is any deoxyribonucleic acid or ribonucleic acid of interest, from any biological sample. The target nucleic acid can be isolated from a sample according to any number of methods well known to those skilled in the art. The category of "nucleic acids" includes but is not limited to DNA, mRNA, cDNA, and cRNA. A target nucleic acid may be genomic in origin or may be made by reverse transcription of the RNA, or second strand cDNA, or RNA transcribed from the double-stranded intermediate, or nucleic acid generated by other means well known by those skilled in the art.

In the presence of nucleic acids containing the exact target sequence, i.e. , when the nucleic acid is complementary to both the capture and counting probes, then the immobilized capture probe hybridizes to the target nucleic acid and the counting probe hybridizes to the target nucleic acid, immediately upstream of the capture probe. Ligation of the capture probe and the counting probe can take place only when there is an exact nucleic acid match. Single-stranded, circular DNA hybridizes to sequences on the 3' end of the counting probe, and copious amounts of DNA are produced in a rolling-circle amplification, according to the invention. The resultant amplification product can be detected and quantified, again in keeping with the present invention, by comparison to a set of reference targets, which are used to generate a reference mathematical formula and curve.

In the presence of non-specific nucleic acids, the immobilized capture probe and the counting probe do not hybridize to the non-specific nucleic acid. Thus, the 3' end of the capture probe is not adjacent to the phosphorylated 5' end of the counting probe; yet such an orientation of the capture and counting probes is necessary for the ligation reaction to occur. In the presence of non-specific nucleic acids, the capture and counting probe are not properly aligned and, hence, the counting probe is not ligated to the capture probe. Without the ligation reaction, the counting probe is eliminated in the subsequent wash step, and neither hybridization to the single-stranded circular DNA nor amplification of single-stranded circular DNA occurs.

In the presence of nucleic acids containing the target nucleic acid sequence but varying by one or a few nucleotides, the immobilized capture probe hybridizes to the nucleic acid, and the counting probe hybridizes to the nucleic acid immediately upstream of the capture probe. In this instance, however, a mismatch occurs at the site of the nucleotide differences. (A "mismatch" occurs when nucleotides within the target nucleic acid do not form a non-covalent interaction with one or more nucleotides in the capture or counting probe.) As noted, ligation of the capture probe and the counting probe can take place only when there is an exact nucleic acid match at the junction of the capture and counting probe. Accordingly, a mismatch at the 5' end of the capture probe or the 3' end of the capture probe interferes with the ligation reaction. Without the ligation reaction, the counting probe is eliminated in the subsequent wash step, and neither hybridization to the single-stranded circular DNA nor amplification of single-stranded circular DNA takes place. Therefore, by designing capture and counting probes such that the nucleotide or nucleotides of interest are located at the junction of the counting and capture probe, one can apply the present invention to the task of detecting as small as a single-nucleotide difference.

Samples from diverse sources contain nucleic acids of interest that are subject to detection, analysis, and/or quantification in accordance with the present invention. Sources for target nucleic acid may be any biological source, including pre-natal and post-mortem samples. Biological sources include but are not limited to viruses, bacteria, parasites, fungi, single cells, body fluids, excrement, tissues. Target nucleic acid can be isolated from a biological source pursuant to convention methodology. Due to the diversity of sources that are accessible, the present invention has a wide variety of applications in medical diagnostics, in the identification of genetic background, in the recognition of drug-resistant strains, in environmental monitoring, in forensics science, and in basic and industrial research.

Thus, the present invention can be utilized to detect the presence of an infectious disease by identifying the relevant pathogen(s). Including bacteria, viruses, parasites, and fungi, inter alia, a varied number pathogens can be identified and the related infectious disease diagnosed by extracting nucleic acid from a sample and identifying the presence or absence of a particular pathogen within the sample, in accordance with the present invention. Illustrative of the pathogens that can be detected in this manner are Escherichia coli, Salmonella, Shigella, Klebsiella, Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis, Mycobacterium aviumintracellulare, Yersinia, Francisella, Pasteurella, Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcus aureus. Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria, Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea, Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis, Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponema palladium, Borrelia burgdorferi, Borrelia recurrent is, Rickettsial pathogens, Nocardia, Acitnomycetes, Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasma capsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candida albicans, Aspergillusfumigautus, Phycomycetes (Rhizopus), Sporothrix schenckii, Chromomycosis, Maduromycosis, human immunodeficiency virus, human T-cell lymphocytotrophic virus, hepatitis viruses (e.g. , Hepatitis B Virus and Hepatitis C Virus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses, orthomyxo viruses, paramyxoviruses, adenoviruses, corona viruses, rhabdo viruses, polio viruses, toga viruses, bunya viruses, arena viruses, rubella viruses, reo viruses, Plasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodium ovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosoma spp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonas spp., Balatidium coli, Wuchereria bancrofti, Toxoplasma spp., Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura, Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp., Pneumocystis carinii, and Necator americanis.

Additionally, this invention can be used to identify the specific strain of a particular pathogen. Identification of the specific strain or variants of a strain is of use in a variety of applications including determining antibiotic resistance such that an effective course of antibiotic treatment can be devised. Amenable to such identification, for example, are vancomycin-resistant Enterococcus faecium, methicillin- resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, multi-drug resistant Mycobacterium tuberculosis, and AZT- resistant human immunodeficiency virus. The present invention also addresses the need for determining genetic predisposition and/or confirming medical diagnosis of genetic disease. By designing capture and counting probes to hybridize with the target nucleic acid of interest, the presence or absence of as little as a single nucleotide mutation can be determined within a nucleic acid pool containing the patient's entire genome. Mutations include insertions, deletions, single base changes, and translocations. Exemplary of such genetic diseases are: 21 hydroxylase deficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome, Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heart disease, single gene diseases, HLA typing, phenylketonuria, sickle cell anemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome, Huntington Disease, autoimmune diseases, lipidosis, obesity defects, hemophilia, inborn errors of metabolism, and diabetes.

Another area of application for this invention relates to screening for the presence or absence of a growing number of sequences, in a target nucleic acid, that are associated with a disease, such as the mutant sequences linked, respectively, to cystic fibrosis. In screening for genetic predisposition and/or to confirm diagnosis of this disease, it is optimal to screen, via a single assay, for all possible mutations within the genome of a subject. The present invention has the capacity, for instance, to screen for every known cystic-fibrosis mutation, in a single sample, by means of a single reaction.

Additionally, the present invention provides for the simultaneous monitoring of the expression levels of a multiplicity of genes in a single reaction. Simultaneous monitoring permits accurate comparisons of gene expression levels and allows for the identification of biological conditions that alter gene expression. Genes of particular interest for expression monitoring include: the HER2 (c-erbB-2/neu) proto-oncogene, in the case of breast cancer; receptor tyrosine kinases (RTKs) that are implicated in the etiology of a number of tumors, including carcinomas of the breast, liver, bladder and pancreas, as well as glioblastomas, sarcomas and squamous carcinomas; and tumor suppressor genes such as the P53 gene and other "marker" genes, e.g. , RAS, MSH2, MLH 1 and BRCA1 . Additional genes of particular interest for expression monitoring are genes involved in the immune response, such as interleukin genes, as well as genes involved in cell adhesion (e.g., the integrins and selectins), apoptosis and signal transduction (e.g. , tyrosine kinases).

Moreover, the present invention can be used to detect a mutant gene that results in oncogenesis. Specifically, the capture probe and the counting probe can be designed such that they hybridize with nucleic acid sequences known to result in aberrant cell growth. Exemplary of the detectable genes involved in oncogenesis are the BRCA1 gene, the p53 gene, the APC gene, Her2/Neu amplification, the Bcr/Abl, K-ras gene, and human papillomavirus Types 1 6 and 1 8.

This invention also may be used to identify an individual genetically. The presence or absence of a set of a multiplicity of nucleic acid sequences that are unique to an individual can be determined in a single reaction, using a single sample. The result is useful in the context of forensics, paternity testing, and other applications that concern individual identification.

Additionally, this invention may be used to determine the presence of specific variants of genes for immunocapable organ donor matching of organ, tissues, and bodily fluids prior to transplantation.

The invention also has a wide variety of food, feed, and agricultural applications, including detection and identification of plant-specific pathogens and veterinary infections, identification of genetic background for plant or animal breeding, and identification of organisms or genes that affect plant or animal growth, health and/or quality. In general, therefore, the present invention has use in clinical, industrial, and research fields.

The present invention is further described by reference to the following, illustrative examples.

Example 1 .

A. Materials

1 . The solid surface used in the inventive method may be glass, plastic, or any other material to which the capture probes can be immobilized.

2. The capture probes are synthetic DNA oligonucleotides (oligos), which are designed to capture specific targets. The 5'-end of the capture probes is modified with active chemical groups, such as biotin or primary amine with a linker. The capture oligos range in size from about 20 nucleotides to about 100 nucleotides. The preferred size of the probe is around 60 to 75 nucleotides. The sequences of the probes are selected such that they are complementary to a conserved region of the target sequences. The melting temperature (Tm) of the probes should be around 50°C to 80°C. 3. The counting probes also are synthetic DNA oligos, designed to count the target molecules. The 5'-end of the oligos are phosphorylated which allows ligation to occur between the capture and the counting oligos. The counting probes contain two major portions: the 5'- portion and the 3'- portion. The 5'- portion of the probes is complementary to a region on the target sequences located immediate up stream of the region of which the capture probe anneals. The sizes of the 5'- portion range from 50 to 70 nucleotides. The Tm values for the annealing should be between 50°C to 80°C. The 3'- portion of the probes is complementary to a portion of the single stranded circular DNA. The sizes of this portion range from 1 8 to 30 nucleotides. The Tm values should be around 50°C to 70°C. The sequences should be unique among the sequences of the single-stranded, circular DNA. 4. The amplifier is a single-stranded, circular DNA molecule, which is a key element in the signal-amplification reaction. The circular DNA may be synthetic or isolated, natural DNA, such as M 1 3 ssDNA or its derivatives, e.g. , M 1 3mp ssDNA. The amplifier should not have strong secondary structures, which may interfere with the efficiency and accuracy of the DNA amplification reaction, and should not have repeat sequences, which may induce template switches, in order that the efficiency of the amplification could be reduced. The sequences complementary to the 3'-end of the counting probes are required for the amplification reactions. 5. The polymerase is another key element of the amplification machinery. Higher processivity and strand-displacement activity of the enzyme are important for the amplification reaction. Escherichia coli DNA ploymerase I, phi29 DNA polymerase, and other DNA polymerases with the mentioned features are preferred DNA polymerases for the amplification reaction.

6. The DNA ligase also is a key factor for the accurate counting of targets. The ligase catalyzes a reaction that covalently connects the counting probes to the capture probes. The number of the counting probes annealed with the capture probes is the same as the number of the targets in a sample. Therefore, the ligation efficiency directly affects the sensitivity and the quantification accuracy of the method. The DNA ligase should be sensitive to mismatches at or nearby the nick site. This feature increases the specificity of the method. T4 DNA ligase and other DNA ligases with the mentioned features are the preferred ligases for this method. 7. Single-stranded nucleic acid binding proteins and other nucleic acid-chaperone proteins stimulate the amplification reaction, thereby increasing the sensitivity of the method. The proteins also increase the rates of the annealing reactions and help to form the most stable nucleic acid hybrid, allowing for greater specificity.

8. Any conventional nucleotide labels, nucleotide analogous and their ligands or detection reagents. These reagents shall be used for detecting and quantifying the amount of DNA synthesized at the end of the amplification reaction. The amount of label is proportionate to the amount of the ligated capture and counting probes, which together represent the amount of target in the sample. The sensitivity of the detection system is a key factor. However, any detection systems may be used to ascertain the presence and quantity of label.

9. The four deoxynucleotides, dATP, dGTP, dCTP and dTTP, DTT, along with salts and reaction buffers.

1 0. Materials for the quantification reference. The quantification reference includes sets of capture probes, counting probes, reference targets, and all other materials described above. The features of the capture probes and counting probes are same as the described above. The reference targets are a group of chemically and/or enzymatically synthetic DNA and/or RNA oligos. The length of the oligos ranges from 40 to 1 00 nucleotides, or longer. The typical length of the oligos is 60 to 80 nucleotides. The Tm values of the oligos shall be in same range as the described above for sample testing probes.

B. Procedures

1 . Immobilization of the capture probes onto a solid surface a. The capture probes is printed onto a glass slide or other solid surface. The capture probes is modified at the 5'-end with primary amine group with a 7-carbons linker. A "DNA chip printer" prints the modified capture probes on the glass slides. The printing variation shall be less than 5%. b. The capture probes shall be immobilized into the wells of a 96- well or 384-well plate. The plates shall be coated with avidin or streptavidin, or with another ligand. The capture probe shall be modified at the 5-end with biotin or another ligand. The spotting variation preferably is less than about 2-5%. Each sample well can be spotted with one capture probe, or multiple probes. 2. Isolation of targets from samples The target DNA or RNA shall be isolated from samples, such as blood, tissue fluids, supernatants of cultures, tissues, cell masses, and any other samples.

3. Hybridization of capture probes and counting probes with the isolated targets The isolated targets shall be incubated with the capture and counting probes at temperature from about 37 °C to 80°C in a mixture with salts, acid-basic buffers, and optional annealing stimulation reagents such as spermidine, nucleic acid chaperone proteins. A typical annealing procedure is as follows: incubate the mixture at 65-70°C for 5 to 1 0 minutes, then, over a period of 1 5 to 30 minutes, gradually cool the mixture to 25 °C. This procedure allows the capture probe to trap its specific targets and allows the counting probe to find and anneal to targets. The annealing of the counting probes to the targets is at a one to one ratio. Each target allows only one counting probe to annealing. This ensures the accuracy of the quantitative measurement. 4. Ligation

A ligase and ATP shall be added into the reaction chamber. Incubation then takes place at about 25 to 37 °C for 5 to 30 minutes. 5. Wash

After the ligation reaction, a wash procedure is applied. Extensive washing is desirable and ensures that extra counting probes, non-specific targets, and the targets themselves are removed. After the wash, only those counting probes covalently linked with the capture probes remain in the reaction.

6. Assembly of DNA polymerization machinery

After the wash, single stranded circular DNA are added to the reaction mixture which shall be incubated at about 65 to 70°C for 5 to 1 0 minutes, then gradually cool down to 25 °C over a period of 1 5 to 30 minutes. This procedure allows the primer, at the 3'-end of the counting probe, to anneal to the single stranded circular DNA. This annealing reaction assembles the DNA amplification machinery.

7. DNA amplification DNA polymerase and a reaction buffer with dNTPs are added into the reaction chambers. A mixture for the DNA amplification is assembled. In the mixture, detectable dNTP or dNTP coupled with a detectable molecules are included such that the DNA products generated at the end of the amplification reaction can be detected and quantified. The reaction mixture shall be incubated at 37°C for about 2 to 6 hours.

8. Detection

The DNA products can be detected and quantified via a procedure based on the type of the labels used in the amplification reaction. C. Quantification and interpretation of results 1 . Establish a reference mathematical formula and curve. Ten different targets, which, as described in the "Materials" section above, also are synthetic DNA or RNA probes, are added into the annealing mixture in the above procedure A 3. Since each of the 1 0 targets was quantified in advance, the copy number of each target is known. Therefore, a reference curve and a mathematical formula of the detection readings versus the copy number can be created. The readings of each sample can then be calculated using this formula and compared with the reference curve. In this way, the readings can be translated into copy numbers. 2. Since the ligation efficiencies are different among DNA templates and RNA templates, if the targets for the reference curve are DNA while the target in a sample is RNA, or the targets are a mixture of RNA and DNA there will be some discrepancy in measurements. These discrepancies can be calibrated, however, because the ligation efficiencies can be measured in advance by use of RNA or DNA templates, of which the copy numbers are known. Example 2. Microarray Preparation

1 . Oligonucleotide Probes Synthesis Oligonucleotide probes were synthesized by MWG Biotechnologies

Inc. with a covalently attachment of amino-linker group to the 5' end of the capture oligonucleotides and a phosphorylation at the 5' end of the counting oligonucleotide. [insert* : What is "MWG"?]

2. Microarray Fabrication (a) Resuspend the amino-modified oligonucleotide probes in distilled H2O at 200 pmol/μl .

(b) (Optional) 1 : 1 dilute each probe in 6xSSC or DMSO spotting solution

(c) Transfer 1 0 μl of each dilution into 384-well micro-plate;

(d) Use Cartesian PS 5500 contact microarrayer to print the samples onto Slides (CSS-1 00 Silyated Slides from TeleChem International Inc., the silylated slides covalently bind ssDNA or dsDNA directly to the surface of a high quality microscope slide via the Schiff base aldehyde- amino chemistry) with Micro Spotting Pins at 25 ° C room temperature, 70% relative humidity, and incubate at same condition for 1 hour. The spots were 200 um in diameter and 500 um in center-to-center spacing. (e) Allow the slides to dry for about 1 2 hours at room temperature (25 ° C) at < 30% relative humidity.

3. Post-Processing to Fix Amino-modified ,

Oligonucleotide Probes to Slide (a) Rinse the slides once in 0.1 % SDS for 2 minutes at room temperature with vigorous agitation to remove the unbound DNA;

(b) Rinse the slides twice in dH2O for 2 minutes at room temperature with vigorous agitation;

(c) Treat the slides in sodium borohydride solution (Dissolve 0.5 g NaBH4 in 1 50 ml PBS , then add 50 ml 1 00% ethanol to reduce bubbling and prepare just prior to use) for 5 minutes at room temperature, to reduce free aldehydes;

(d) Rinse the slides twice in dH2O for 1 minute at 37°C;

(e) Dried the slides in air. 4. Quality Control

Fluorescent nucleic acid stain (dimeric cyanine dye TOTO-3 from Molecular Probes) was used to check the uniformity of spotting features according the product protocol:

(a) Dilute the stain 10,000-fold in TE bufferd O mM Tris, 1 mM EDTA, pH 8.0);

(b) Cover the microarray with diluted stain and incubate at room temperature for 3 minutes.

(c) Wash the microarray three times with TE buffer.

(d) Spin-dry the microarray. (e) Scan the microarray using ScanArray 4000B scanner(Axon Inc.)

Hybridization and signature amplification

1 . Annealing the targeting DNA (fragment) with counting oligonucleotide and capture oligonucleotide (a) Dilute counting DNA oligonucleotide, at a final concentration of

^" 1 pm/μl, and target DNA into annealing buffer (50mM Tris.HCI pH7.5, 75mM KCI) to make the hybridization mixture;

(b) Cover microarray with 1 0 μl hybridization mixture; (c) Put the slide in hybridization chamber (CMT hybridization chamber from Corning Inc). Add 50 μl annealing buffer in the inner holes of hybridization chamber and close up the chamber.

(d) Incubate hybridization chamber in 82°C water bath for 2 minutes, and then transfer the chamber to a water bath at 53°C, for 30 minutes; (e) Gradually cool the chamber (in water bath) to 25°C in 30 minutes. 2. Ligation

(a) Open the cover of the chamber and absorb the annealing buffer;

(b) Add 1 0 μl of ligation mixture (2 units/ul Promega T4 DNA ligase in 1 x buffer provided with enzyme) to the microarray; Incubated at 25°C room temperature for about 1 5 minutes to 2 hours (depends on the usage of different batch of enzyme) to ligase capture sequence and detecting sequence;

(c) Wash the slide with 1 x Annealing buffer for 1 minute x 5 times at room temperature; 3. Annealing the single-stranded circular DNA to counting probe

(a) Dilute the single-stranded circular DNA (M 1 3mp1 9 strand DNA from Invitrogen) to a final concentration of 0.025 μg/μl in Annealing Buffer;

(b) Add 1 0 μl the dilution to microarray and then put the slide into hybridization chamber; (c) Incubate hybridization in 53°C water bath for 30 minutes and then gradually cool down to 25°C in 30 minutes;

(d) Wash the slide in wash buffer (50 mM Tris.HCI pH7.5, 1 0 mM MgCI2, 2 mM DTT) for 1 minute x 5 times;

4. Extension Reaction (a) Cover the microarray with 10 ul Extension Reaction Mixture; (b) Place the slide in hybridization chamber and incubate in 37°C for ~ 4 hours;

(c) Wash the slide in 30 ml TE buffer for 5 minutes x 3 at 25°C room temperature, then dried slide for scanning.

Extension Reaction Mixture

1 0x Buff er(provided with enzyme) 1 .0 μl

DNA Polymerase I (Promega M2051 9.2 μg/μl) 1 .0 μl l OO mM dATP 0.2 μl l OO mM dGTP 0.2 μl l OO mM dTTP 0.2 μl

1 0 mM dCTP 0.2 μl

Cyanine 3-dCTP(25nM/25μl,NEN) 2.0 μl dH2O 5.2 μl

Image detection and data analysis

ScanArray 4000B Laser scanner from Axon Inc was used to detect the signature intensity of incorporated Cy3-dCTP at 300v PMT, at 5 μm resolution. The GenePix Pro 3.0 Microarray Acquisition & Analysis Software package was employed for data analysis.

Claims

WHAT IS CLAIMED IS:

1 . A method for analyzing nucleic acids, comprising (a) hybridizing a target nucleic acid to a capture probe, (b) hybridizing a counting probe to the target nucleic acid, (c) ligating the capture probe to the counting probe, and (d) hybridizing a single-stranded, circular DNA to counting probe, and then (e) amplifying said circular DNA, wherein said amplification is sufficient for detecting the presence of target nucleic acid.

2. The method according to claim 1 , wherein the target nucleic acid is DNA, RNA, or cDNA.

3. The method according to claim 1 , wherein the target nucleic acid is from a bacterium, a virus, a parasite, or a fungus.

4. The method according to claim 1 , wherein the target nucleic acid sample is taken from body fluid, tissue, or feces.

5. The method according to claim 4, wherein the target nucleic acid sample is taken from blood, saliva, urine, or sputum.

6. The method according to claim 1 , wherein the capture probe is complementary to a nucleic acid sequence from a bacterium, a virus, a parasite, or a fungus.

7. The method according to claim 1 , further comprising determining the quantity of target nucleic acid present by comparing the quantity of amplification product from step (e) to at least one known quantity of nucleic acid standard.

8. A method for analyzing a target nucleic acid, comprising:

(a) hybridizing a target nucleic acid to a capture probe and (b) hybridizing a counting probe to the target nucleic acid, such that a hybrid comprised of capture probe, counting probe, and target probe is formed; (c) exposing said hybrid to a ligase and then (d) washing away counting probe and nucleic acid not covalently linked to said capture probe; then (e) adding a single-stranded, circular DNA to said capture probe; (f) adding to said capture probe a nucleic acid polymerase that can amplify single-stranded circular DNA; and then (g) ascertaining whether amplified circular DNA is present or absent.

9. A molecular assemblage comprised of (i) a capture probe, (ii) a counting probe, and (iii) a single-stranded, circular DNA segment, wherein said circular DNA segment is contiguous with said counting probe.

1 0. The molecular assemblage according to claim 9, wherein the target nucleic acid is DNA, RNA, or cDNA.

1 1 . The molecular assemblage according to claim 9, wherein the target nucleic acid is from a bacterium, virus, parasite, or fungus.

1 2. The molecular assemblage according to claim 9, wherein the capture probe is complementary to a nucleic acid sequence from a bacterium, a virus, a parasite, or a fungus.

1 3. The molecular assemblage according to claim 9, wherein the target nucleic acid is from body fluid, tissue, or feces.