KR20120046018A - Real time pcr detection of single nucleotide polymorphisms - Google Patents

Abstract

PURPOSE: A real-time PCR detection method for single nucleotide polymorphism is provided to ensure quick and accurate detection. CONSTITUTION: A real-time PCR detection method for polymorphism in a target DNA comprises: a step of providing a test sample to test the target DNA with polymorphism; a step of providing amplification primer and annealing a first primer to the upstream of location of polymorphism and a second primer to the downstream of location of polymorphism; a step of proving a probe containing detectable label, DNA, and RNA nucleic acid sequence.

Description

Real-time PCR detection of single nucleotide polymorphisms {Real-time PCR detection of single nucleotide polymorphisms}

This patent application claims priority from U.S. Provisional Patent Application No. 61 / 390,701, filed October 7, 2010, and U.S. Patent Application No. 13 / 158,593, filed June 13, 2011, The contents of which are incorporated herein by reference in their entirety.

This disclosure describes the real-time PCR detection of single nucleotide polymorphism (SNP) using a SNP-specific CataCleave (TM) probe.

The completion of the Human Genome Project (HGP) provides a better understanding of the genetic diversity within a particular population of humans and how diversity relates to the onset or predisposition of genetic disease . For example, a single nucleotide difference between individuals, referred to as a single nucleotide polymorphism (SNP), is a phenotypic phenotype that, in some circumstances, can predict who will be suffering from a particular disease, or who will respond to a particular treatment for the disease This may be the cause of the dramatic difference in The scope of pharmacogenomics is a rapid approach in which medical treatment is tailored to the patient's genetic make-up. Rapid and reliable detection of genetic mutations in the patient ' s DNA can provide guidance in the selection of a physician ' s clinical diagnosis and medical treatment. This is particularly true in oncology, where the presence of discrete mutations in the coding region of the oncogene can be a strong predictor of cancer susceptibility and prognosis.

For example, genetic testing for the presence of deleterious mutations in the BRCA tumor suppressor gene is now common in most medical practices. The deleterious BRCA-1 mutation may significantly increase the risk of developing breast and ovarian cancer in women and risk of developing cervical, uterine, pancreatic, and colorectal cancer at lower ages (premenopausal). The deleterious BRCA2 mutation may additionally increase the risk of pancreatic cancer, stomach cancer, gallbladder cancer and bile duct cancer, and melanoma. Mutations in several other genes, including TP53 , PTEN , STK11 / LKB1 , CDH1 , CHEK2 , ATM , MLH1 , and MSH2 have been associated with hereditary breast tumors and / or ovarian tumors.

With the advent of nucleic acid amplification, even small single molecules of any DNA sequence can be replicated a sufficient number of times to enable SNP sequencing. SNPs can be used for DNA sequencing, fluorescent probe detection, mass spectrometry or DNA microarray hybridization (see, for example, U.S. Patent Nos. 5,885,775; 6,368,799 And the like). Many of these methods are inadequate despite high application throughput due to overall poor sensitivity, cost, time spending or the need for post-PCR processing. Existing SNP detection methods also exhibit unacceptably high levels of false positive and / or false negative results.

For the reasons stated above, there is a need in the art for an unmet need for accurate real-time detection of a SNP, which is performed simultaneously with DNA amplification.

Methods and kits for rapid detection of SNPs during real-time PCR using specific probes comprising DNA and RNA sequences are disclosed. The method ensures that, in a cost effective and reliable manner, it promotes high throughput detection of one or more SNPs on a single PCR fragment.

In one embodiment, providing a sample to be tested for the presence of a target DNA having a polymorphism, wherein a first amplification primer anneals upstream of the position of the polymorphism and a second amplification primer anneals to the polymorphic position Providing a pair of amplification primers capable of annealing to the target DNA, annealing the amplification primer downstream of the amplification primer pair, and providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence , The RNA nucleic acid sequence of the probe is entirely complementary to a selected region of the target DNA sequence comprising the polymorphism and the DNA nucleic acid sequence of the probe is substantially complementary to the DNA sequence adjacent to the selected region of the target DNA sequence wherein the RNA sequence in the probe is complementary to a complementary DNA sequence in a PCR fragment comprising the polymorphism, : Amplification polymerase activity, amplification buffer, under conditions that allow formation of DNA heterodimers (heteroduplex); Amplifying a PCR fragment between the first and second amplification primers in the presence of RNase H activity and a probe and detecting a real increase in signal emission from the label on the probe, Lt; RTI ID = 0.0 > polymorphism < / RTI > in the target DNA.

In one embodiment, the real-time increase in signal emission from the label on the probe is due to RNase H cleavage of the probe's RNA sequence in the RNA: DNA heterodimer double strand.

In another embodiment, there is provided a method of detecting a polymorphism comprising: providing a sample to be tested for the presence of a target DNA having polymorphism; annealing the first amplification primer upstream of the polymorphic position; and annealing the second amplification primer downstream of the polymorphic position Providing a probe comprising an amplifiable primer pair capable of annealing to said target DNA, a detectable label, and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of said probe is at said polymorphic position Wherein the probe nucleic acid sequence is substantially complementary to a selected portion of a target DNA sequence comprising a wild type DNA sequence and wherein the DNA nucleic acid sequence of the probe is substantially complementary to a DNA sequence adjacent to a selected portion of the target DNA sequence, Wherein the RNA sequence in the first region is a complementary DNA sequence in a PCR fragment comprising a polymorphism and a second region capable of forming an RNA: DNA heterozygous double strand Under, the amplifying polymerase activity, an amplification buffer; Amplifying a PCR fragment between said first and second amplification primers in the presence of RNase H activity and a probe and detecting a real-time decrease in signal emission from the label on said probe, Lt; RTI ID = 0.0 > polymorphism < / RTI > in the target DNA.

In yet another embodiment, there is provided a method comprising: providing an RNA target; providing an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer anneals upstream of the polymorphic sequence position, Annealing downstream of the polymorphic sequence position; Providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence substantially complementary to the cDNA of the RNA target, wherein the RNA nucleic acid sequence of the probe is in a corresponding cDNA sequence of the suspected SNP sequence position A step that is completely complementary; In the presence of a reverse transcriptase activity, an amplified polymerase activity, a reverse transcriptase-PCR buffer, a site-specific RNase H activity, and a probe, and wherein the RNA sequence in the probe is an RT-PCR DNA fragment Amplifying a reverse transcriptase-PCR fragment between the first and second amplification primers under conditions that allow formation of a complementary DNA sequence and an RNA: DNA heterozygous double strand within the first amplification primer; And detecting a real-time increase in signal emission from the label on the probe, wherein the increase in signal indicates the presence of a polymorphism in the cDNA of the RNA target. do.

In one embodiment, the real-time increase in signal emission from the label on the probe is due to RNase H cleavage of the probe's RNA sequence in the RNA: DNA heterodimer double strand.

Providing a sample to be tested for an RNA target having a polymorphism, providing an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer is located upstream of the polymorphic position Annealing the second amplification primer and annealing the second amplification primer downstream of the polymorphic position, providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is in a wild type Wherein the DNA nucleotide sequence of the probe is substantially complementary to the DNA sequence adjacent to the selected site of the cDNA, the reverse transcriptase activity, the amplified polymerase activity, the reverse transcriptase In the presence of a PCR buffer, RNase H activity and a probe, and wherein the RNA sequence in said probe is in the presence of wild-type DNA Amplifying a reverse transcriptase-PCR fragment between said first and second amplification primers under conditions that allow formation of a double strand of RNA: DNA heterozygosity with a complementary sequence in an RT-PCR DNA fragment comprising a sequence, A method for real-time detection of polymorphism in an RNA target is disclosed, comprising detecting a real-time decrease in signal emission from a label on a probe, wherein the reduction in signal indicates the presence of a polymorphism in the RNA target.

In yet another embodiment, the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position, an amplification primer pair capable of annealing to the target DNA, a detectable label, and a DNA And the RNA nucleic acid sequence of the probe is completely complementary to the selected region of the target DNA sequence comprising the polymorphism and the DNA nucleic acid sequence of the probe is a DNA sequence adjacent to the selected region of the target DNA sequence A probe that is substantially complementary to the amplification polymerase activity, an amplification buffer; And RNase H activity, is disclosed herein.

In yet another embodiment, the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position, an amplification primer pair capable of annealing to the target DNA, a detectable label, and a DNA And a probe comprising an RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected portion of a target DNA sequence comprising a wild-type DNA sequence at a polymorphic position, and the DNA nucleic acid sequence of the probe is selected A probe that is substantially complementary to the DNA sequence adjacent to the site, and amplified polymerase activity, amplification buffer; And RNase H activity, are disclosed herein.

In yet another embodiment, the first amplification primer anneals upstream of the polymorphic sequence position and the second amplification primer anneals downstream of the polymorphic sequence position, wherein the amplification annealable to the cDNA of the RNA target A probe comprising a primer pair, a detectable label, and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to the selected site of the cDNA comprising the polymorphism, and the DNA nucleic acid sequence of the probe comprises a selected region A reverse transcriptase activity, an amplified polymerase activity, a reverse transcriptase-PCR buffer; Lt; / RTI > and RNase < RTI ID = 0.0 > H < / RTI > activity.

In another embodiment, the first amplification primer anneals upstream of the polymorphic sequence position and the second amplification primer anneals downstream of the polymorphic sequence position, an amplification primer pair capable of annealing to the cDNA of the RNA target, Wherein the probe nucleic acid sequence is fully complementary to a selected region of a cDNA comprising a wild-type DNA sequence at a polymorphic position, and wherein the DNA nucleic acid sequence of the probe is selected And a reverse transcriptase activity, an amplified polymerase activity, a reverse transcriptase-PCR buffer; Lt; / RTI > and RNase < RTI ID = 0.0 > H < / RTI > activity.

The polymorphism may be a single nucleotide polymorphism (SNP).

The target DNA may be a genomic DNA. The target RNA may be a genomic RNA or an mRNA transcript.

The DNA and RNA sequences of the probes can be covalently linked.

The detectable label on the probe may be a fluorescent label, such as a FRET pair. The PCR fragment or probe may be bound on a solid support.

The amplified polymerase activity may be a thermostable DNA polymerase and the site-specific RNase H activity may be a heat-stable RNase H activity or a hot-start thermostable RNase H activity.

In certain embodiments, the reverse transcriptase and amplified polymerase activities are found in the same molecule.

The embodiments described above have a number of advantages, including the ability to detect the presence of a SNP in real time on a target nucleic acid. The detection method is fast, accurate, and suitable for high throughput applications. A convenient, easy to use, reliable diagnostic kit for SNP detection in different genetic loci is also disclosed.

Skilled artisans will appreciate that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Figure 1 in the Salmonella (Salmonella) invA gene showing the isothermal detection (isothermal detection) of the wild-type target. An increase in fluorescence intensity is observed when an exact pairing (wild-type probe: wild-type target) is observed, but an increase in fluorescence intensity is not observed when an inaccurate pairing (wild-type probe: SNP-containing target).
Figure 2 shows the isothermal detection of a SNP target (change from T to G base) in Salmonella invA gene. An increase in fluorescence intensity was observed when correctly matched (SNP probe: SNP target), but no increase in fluorescence intensity was observed when inaccurate pairing (SNP probe: wild type target).
Figure 3 shows multiplex real-time detection of a wild-type target in Salmonella invA gene. An increase in fluorescence intensity is observed in the presence of a homozygous wild-type target or heterozygous wild-type SNP target. In the presence of a homozygous SNP target, no increase in fluorescence intensity was observed.
Figure 4 shows multiplex real-time detection of SNP targets in Salmonella invA gene using a SNP sensing probe. In the presence of homozygous SNP target or heterozygous SNP-wild type target, an increase in fluorescence intensity is observed. In the presence of a homozygous wild-type target, no increase in fluorescence intensity is observed.
Figure 5 shows the isothermal detection of A1 b casein. An increase in fluorescence intensity is observed when correctly matched (A1 probe: A1 target), but no increase in fluorescence intensity is observed when inaccurate pairing (A1 probe: A2 target).
Figure 6 shows the isothermal detection of A2 b casein. Accurately matched A2 probe: An increase in fluorescence intensity was observed at A2 target, but no increase in fluorescence intensity was observed for A2 probe: A1 target that was incorrectly matched.
Figure 7 shows multiplex real-time detection of A1 b casein using an A1 sensing probe. An increase in fluorescence intensity is observed in the presence of homozygous A1 target or heterozygous A1-A2 target. The increase in fluorescence intensity is not observed in the presence of a homozygous A2 target.
Figure 8 shows multiplex real-time detection of A2 b casein using an A2 sensing probe. An increase in fluorescence intensity is observed in the presence of homozygous A2 target or heterozygous A1-A2 target. The increase in fluorescence intensity is not observed in the presence of homozygous A1 target.
Figure 9 is a table describing all PCR primers, probes, and targets used in the figures and examples. The locations of the SNPs are underlined.
FIG. 10 is a graph showing the distribution of the < RTI ID = 0.0 > Pyrococcus & furiosis ), Pyrococcus horikoshi), Thermo Lactococcus coder Karen sheath (Thermococcus kodakarensis), Douce (Archeoglobus booth Pro extracted with ahreuke Ogle profundus), solve booth to display ahreuke Ogle (Archeoglobus fulgidis), Thermococcus celer (Thermococcus celer) and Thermococcus litoralis (Thermococcus litoralis ) RNase HII polypeptide sequences.
Fig. 11 is a graph showing the distribution of Haemophilus influenzae influenzae , Thermus thermus thermophilis ) , Thermus aquaticus ( Thermus acquaticus , Salmonella enterica enterica ) and Agrobacterium tumefaciens RNase HI polypeptide sequences.

The practice of the present invention employs conventional molecular biology techniques within the skill of the art, unless otherwise specified. Such techniques are well known to those skilled in the art and are fully described in the literature. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y., including all supplemental data by Ausubel et al. (1987-2008); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. This specification also provides definitions of terms to aid in the interpretation of the present disclosure and the claims. If this definition does not match the definitions elsewhere, the definitions given in this application will prevail.

The term "polymorphism" refers to the presence of two or more alternative genomic sequences or alleles between different genomes or individuals, or among said genomes or individuals.

The term "polymorphic" refers to a state in which two or more variants of a particular genomic sequence are found within a population.

The term "polymorphic site" means the locus at which the mutation occurs. Polymorphic sites generally have two or more alleles, each occurring at a significant frequency within the selected population. A polymorphic locus can be as small as one base pair, in this case referred to as a single nucleotide polymorphism (SNP). The first identified allele form is optionally designated as a reference, wild-type, common, or major form, and allelic forms may be referred to as alternative, minor, It is named as a rare or variant allele.

The term "genotype" refers to a description of alleles of a gene contained in an individual or sample.

The term "single nucleotide polymorphism" ("SNP") refers to a single nucleotide site that differs between alleles. A single nucleotide may be altered (substituted), removed (deleted), or added (inserted) to the polynucleotide sequence. Insertion or deletion SNPs can result in translational frameshifts. A single nucleotide polymorphism can belong to a coding sequence of a gene, a non-coding region of a gene, or an intergenic region between genes. SNPs in the coding sequence will not necessarily change the amino acid sequence of the protein produced due to degeneracy of the genetic code. SNPs in which both forms result in the same polypeptide sequence are termed synonymous (sometimes referred to as silent mutations), but when different polypeptide sequences are produced they are nonsynonymous. Non-kinetic changes may be missense or nonsense, and mis-sense changes produce different amino acids, while non-sense changes result in premature stop codons. A "functional SNP" is a SNP that causes a change in gene expression or expression or function of a gene product, and thus is mostly predictive of possible clinical phenotypes. Changes in gene function by functional SNPs may include changes in the encoded polypeptide, mRNA stability, changes in transcription factors or translational factor binding properties to DNA or RNA, and the like. SNPs that are not in the protein-coding region can display results for gene splicing, transcription factor binding, or sequences of non-coding RNAs.

According to one embodiment, the skilled artisan will understand that SNPs have two alternative alleles, each corresponding to a nucleotide that may be present in the chromosome. Thus, the SNP is characterized by two nucleotides of four nucleotides (A, C, G, T). An example of this would be a SNP with alleles C or alleles T at a given position on each chromosome. This is expressed as C> T or C / T. More commonly occurring alleles are initially displayed (in this case, C) and are called multiple, common, or wild-type alleles. Alternative alleles that occur less frequently (in this case, T) are called prime, rare, or variant alleles. The wild-type and variant alleles may be referred to as general alleles and rare alleles, respectively. Since a person is a diploid organism, meaning that each chromosome exists in two copies, each individual has two alleles in one SNP. These alleles can be two copies of the same allele (CC or TT) or different alleles (CT). The CC, CT and TT are called genotypes. Of these, CC and TT are characterized by having two copies of the same allele and are referred to as homozygous genotypes. The genotype CT has heterozygous genotypes with different alleles on each chromosome. Individuals with a homozygous or heterozygous genotype are referred to as homozygous and heterozygous, respectively.

SNP Choice of

One embodiment provides a novel method for detecting one or more SNPs in any target nucleic acid sequence. The determination of the location of the SNP in the target gene is very easily performed by referring to the biological information database for the SNP. dbSNP is a SNP database provided by the National Center for Biotechnology Information (NCBI). SNPedia is a wiki-style database provided by a merged organization. HGVbaseG2P allows visual querying of relevant summary-level related data, whereas OMIM database documents the association between polymorphism and disease, for example.

Useful information on SNPs can also be found in the International HapMap Project, which identifies genotypes of one informative SNP at about 5 kb across the human genome. In order to determine the general pattern of human DNA sequence variation (haplotype) and make this information freely available in the public domain, genotypes of populations with ancestors derived from Niger, Europe and China / Japan were analyzed . This information will facilitate the discovery of sequence variants affecting common disease and pharmacological responses. The creation of a human half map is an important step for personalized medicine.

For genotype analysis Primer  Selection

Once the gene and related SNPs are selected, primer oligonucleotides and probes are prepared for genotyping the target nucleic acid sequence.

The term "target DNA or target RNA" or "target nucleic acid" or "target nucleic acid sequence" Refers to the region of the nucleic acid. The target nucleic acid sequence is used as a template for amplification in a PCR reaction and a reverse transcriptase-PCR reaction. The target nucleic acid sequence can include both natural and synthetic molecules. Exemplary nucleic acid sequences include, but are not limited to, genomic DNA or genomic RNA.

As used herein, the term "nucleic acid" refers to an oligonucleotide or polynucleotide, which oligonucleotide or polynucleotide may be modified or may comprise a modified base. Oligonucleotides are single-stranded nucleotide polymers, comprising 2 to 60 nucleotides. A polynucleotide is a nucleotide polymer comprising two or more nucleotides. The polynucleotide is a double-stranded DNA comprising an annealed oligonucleotide, wherein the second strand is an oligonucleotide having a reverse complement sequence of the first oligonucleotide, a single-stranded nucleic acid polymer comprising deoxythymine, Single stranded RNA, double stranded RNA or RNA / DNA heteroduplex. The nucleic acid may be a nucleic acid obtained from a subcellular organelle such as genomic DNA, cDNA, hnRNA, snRNA, mRNA, rRNA, tRNA, fragmented nucleic acid, mitochondria or salt lobes, But are not limited to, nucleic acids obtained from microorganisms, DNA or RNA viruses. The nucleic acid can be composed of a mixture of different sugar moieties, such as, for example, in the case of RNA and DNA, a single form of a sugar moiety, or, for example, in the case of RNA / DNA chimera have.

As used herein, the term "oligonucleotide" is used interchangeably with "primer" or "polynucleotide". The term "primer" refers to an oligonucleotide that acts as a starting point for DNA synthesis in a PCR reaction. The primer is typically about 14 to about 35 nucleotides in length and hybridizes to a complementary region of the target sequence.

Oligonucleotides are synthesized and prepared by any suitable method known in the art (such as chemical synthesis). Oligonucleotides are also conveniently available through commercial sources. Skilled artisans will be able to readily optimize and identify primers flanking the target polymorphic site in a PCR reaction. Commercially available primers can be used to amplify a particular gene of interest for a particular SNP. A number of computer programs (e.g., Primer-Express) can be readily used to design optimal primer sets. It will be apparent to those skilled in the art that primers and probes based on nucleic acid information provided (or available by the public as a registration number) can be prepared accordingly.

The terms " annealing "and" hybridization "are used interchangeably and refer to the base pair interaction of a nucleic acid with another nucleic acid, resulting in a form of double strand, triple strand, or other higher order structure. In certain embodiments, the primary interactions are base-specific interactions, such as A / T and G / C, by Watson / Crick and Hoogsteen-type hydrogen bonding . In certain embodiments, base-stacking and hydrophobic interactions can also contribute to double-strand stability. "Substantially complimentary" refers to two nucleic acid strands of a sequence sufficiently anneal to form a stable double strand.

Those skilled in the art will know how to design a PCR primer that is placed on the side of the target polymorphic site. Synthetic oligonucleotides are 20 to 26 base pairs in length, generally having a melting point (T _M ) of about 55 degrees. The flanking sequence for primer design can be found in an allocation file produced by the International HalfMap project. This file contains abundant information for each SNP, including the NCBI-masked sequence of 100 bp NCBI for the observed alleles and the preparation of each side-chain nucleic acid template.

In some embodiments, the sample comprises a purified nucleic acid template (e. G., MRNA, rRNA, and mixtures thereof). RNA extraction and purification procedures from samples are well known in the art. For example, RNA can be isolated from cells using TRIzol ™ reagent (Invitrogen) extraction. The amount and quality of RNA is then determined, for example, using a Nanodrop ^TM spectrophotometer and an Agilent 2100 bioanalyzer.

In another embodiment, the sample comprises a lysis buffer having a pH of from about 6 to about 9, a zwitterionic detergent having a concentration of from about 0.125% to about 2%, from about 0.3 to about 2.5 mg / mL (About 1 mg / mL), such as azide and proteinase K, which have a concentration of about 1 mg / ml of the cell lysate. After incubation at 55 캜 for 15 minutes, the proteinase K was inactivated at 95 캜 for 10 minutes, and the protein, which is compatible with high efficiency PCR or reverse transcription PCR analysis, To obtain a substantially removed lysate.

In one embodiment, the 1 x dissolution reagent comprises 12.5 mM tris acetate or Tris-HCl or HEPES (4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid) (pH = 7-8), 0.25 % (w / v) CHAPS, 0.3125 mg / ml sodium azide and 1 mg / mL protinase K.

As used herein, the term "lysate" refers to a liquid phase comprising dissolved cellular debris and nucleic acid.

As used herein, the term " substantially protein free "refers to a lysate wherein most of the protein is inactivated by proteolysis by protease. The protease may include a proteinase K. Addition of Proteinase K during cell lysis rapidly inactivates the nuclease, which can degrade the target nucleic acid when not inactivated. The term "protein substantially removed" may or may not be treated to remove the inactivated protein.

As used herein, the term "cell" refers to prokaryotic or eukaryotic cells.

In one embodiment, the term "cells" refers to microorganisms, including, but not limited to, gram-positive bacteria, gram-negative bacteria, acid-fast bacteria, etc. In certain embodiments, "Cell" can be collected using swap sampling of the surface. In another embodiment, the "cell" refers to pathogenic organisms.

In other embodiments, the sample comprises a viral nucleic acid, such as a retroviral nucleic acid. In certain embodiments, the sample may contain a lentiviral nucleic acid, such as HIV-1 or HIV-2.

, "Zwitterion detergent (zwitterionic detergent)" are CHAPS, CHAPSO and bar derivative (bine derivative), as used herein, e.g., preferably a trade name Zwittergent ^® (Calbiochem, San Diego, CA) and Anzergent ^® ( Such as but not limited to sulfobines, which are commercially available under the trade designation (e.g., Anatrace, Inc. Maumee, Ohio), exhibiting amphoteric characteristics (e.g., Refers to a detergent that does not have electrophoretic mobility and does not bind to the ion exchange resin and destroys protein-protein interactions.

In one embodiment, the amphoteric ionic detergent has the following structure:

CHAPS (CAS number: 75621-03-3; available from SIGMA-ALDRICH, product number C3023-1G, which is abbreviation of 3 - [(3-colamidopropyl) dimethylammonio] (Described in more detail in U.S. Patent No. 4,372,888).

In a further embodiment, CHAPS is present at a concentration of about 0.125% to about 2% weight / volume (w / v) of the total composition. In a further embodiment, CHAPS is present at a concentration of about 0.25% to about 1% w / v of the total composition. In another embodiment, the CHAPS is present in a concentration of from about 0.4% to about 0.7% w / v of the total composition.

In another embodiment, the lysis buffer may comprise other non-ionic detergents such as Nonidet, Tween or Triton X-100.

As used herein, the term "lysis buffer" refers to a composition capable of effectively maintaining a pH value between 6 and 9, with a pKa of about 6 to about 9 at 25 ° C. The buffers described herein are generally physiologically compatible buffers that are compatible with the function of the enzyme activity and enable the biological macromolecules to maintain their original physical and biochemical functions.

Examples of buffers to be added to the lysis buffer are HEPES ((4- (2-hydroxyethyl) -1-piperazinethanesulfonic acid), MOPS (3- (N-morpholino) (Hydroxymethyl) methylglycine acid (Tricine), tris (hydroxymethyl) methylamine acid (Tris), piperazine-N, N'-bis (2-ethanesulfonic acid) And acetate or phosphate containing buffers (K ₂ HPO ₄ , KH ₂ PO ₄ , Na ₂ HPO ₄ , NaH ₂ PO ₄ ), and the like.

The term "azide ", as used herein, is represented by the formula -N ₃ . In one embodiment, the azide is sodium azide NaN ₃ (CAS No. 26628-22-8, available from SIGMA-ALDRICH, product number: S2002-25G), which acts as a common fungicide.

As used herein, the term "protease" is an enzyme that hydrolyzes peptide bonds (with protease activity). Proteases are also referred to as, for example, peptidases, proteinases, peptide hydrolases, or proteolytic enzymes. The protease for use in accordance with the present invention may be an endo-type (endopeptidase) internally acting on the polypeptide chain. In one embodiment, the protease is a serine protease, proteinase K (EC 3.4.21.64, available from Roche Applied Sciences, recombinant proteinase K 50 U / ml ( Pichia derived from pastoris) Cat. No. 03 115 887 001).

Proteinase K is used to digest proteins and remove contaminants from nucleic acid preparations. The addition of Proteinase K to the nucleic acid agent rapidly inactivates the nuclease which is capable of decomposing DNA or RNA in the tablet when not inactivated. The enzyme is also active in the presence of a chemical denaturing protein and is highly suitable for the present application since it can be inactivated upon treatment at a temperature of about 95 DEG C for about 10 minutes.

In one embodiment, solubility of gram-positive and gram-negative bacteria, such as Listeria Salmonella and E. Coli, is determined using a lysis reagent comprising Proteinase K (1 mg / ml) need. Proteins in cell lysates can be digested by treating proteinase K at 55 ° C for 15 minutes followed by inactivation of proteinase K at 95 ° C for 10 minutes. After cooling, the lysate from which the protein is substantially removed is suitable for high-efficiency PCR amplification.

With or without Proteinase K, the lysis reagent can be a trypsin, chymotrypsin, elastase, subtilisin, streptogrisin, May include serine proteases such as thermotase, aqualysin, plasmin, cucumisin, or carboxypeptidase A, D, C, or Y. In some embodiments, In addition to serine proteases, the lysing solution may be a cysteine protease such as papain, calpain, or clostripain; Acid proteases such as pepsin, chymosin, or cathepsin; B, E / H, M, or N-terminal amino acid residues in the amino acid sequence of SEQ ID NO: T, or U. The term " metalloprotease " Proteinase K is stable over a wide pH range (pH 4.0-10.0) and is stable in buffers containing an amphoteric detergent.

target  Nucleic acid sequence PCR  Amplification

After primers are prepared, nucleic acid amplification can be performed using polymerase chain reaction (PCR), nucleic acid sequence based amplification (NASBA), ligase chain reaction (LCR) But not limited to, rolling circle amplification (RCA). Polymerase chain reaction (PCR) is the most commonly used method for amplifying specific target DNA sequences.

Polymerase chain reaction or PCR generally refers to a method of amplifying a desired nucleotide sequence in vitro. Generally, the PCR method comprises introducing into the reaction mixture containing the desired target sequence (s) a molar excess of two or more extendable oligonucleotide primers, the primers being complementary to the opposite strand of the double stranded target sequence . The reaction mixture is subjected to a thermal cycling program in the presence of a DNA polymerase to amplify the desired target sequence to which the DNA primer is attached on the side.

PCR techniques are described in PCR: A Practical Approach, MJ McPherson et al., IRL Press (1991), PCR Protocols: A Guide to Methods and Applications, Innis et al., Academic Press Erlich, Stockton Press (1989). PCR is also described in U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965, 188; 4,889,818; 5,075,216; 5,079,352; 5,104,792; 5,023,171; 5,091,310; And 5,066, 584, each of which is incorporated herein by reference.

The term "sample" refers to any material comprising a nucleic acid material.

As used herein, the term "PCR fragment" or "reverse transcriptase-PCR fragment" or "amplicon" refers to a polynucleotide molecule produced as a result of amplification of a particular target nucleic acid (Or collectively, a plurality of molecules). PCR fragments are usually, but not always, DNA PCR fragments. The PCR fragment may be single-stranded or double-stranded, or it may be a mixture of these with any concentration ratio. The PCR fragment or RT-PCT may be from about 100 to about 500 nt or longer.

"Buffer" is a compound added to an amplification reaction that alters the stability, activity, and / or longevity of one or more components of an amplification reaction by modulating the pH of the amplification reaction. The buffering agent of the present invention is compatible with PCR amplification and site-specific RNase H cleavage activity. Some buffering agents are well known in the art and include tris, tricine, MOPS (3- (N-morpholino) propanesulfonic acid), and HEPES (4- (2- hydroxyethyl) -1-piperazine ethanesulfonic acid ), But are not limited thereto. In addition, the PCR buffer generally contains up to about 70 mM KCl and about 1.5 mM or more MgCl ₂ , about 50-200 mM each of the respective nucleotide dATP, dCTP, dGTP and dTTP. The buffers of the present invention can contain efficient reverse transcriptase-PCR or additive materials to optimize the PCR reaction.

As used herein, the term "nucleotide" refers to a sugar, such as ribose, arabinose, xylose and pyranose, and sugar analogs thereof, Refers to a compound comprising a nucleotide base linked to a C-1 ' carbon. The term nucleotide also includes nucleotide analogs. The sugar may be substituted or unsubstituted. Substituted ribose sugars, one or more of the carbon atoms, for example, 2'-carbon atom is the same or different, Cl, F, -R, -OR , -NR 2 ( wherein R a each is independently H, C ₁ -C ₆ alkyl or C ₅ -C ₁₄ aryl is a), or one including the halogen group will be substituted with one or more of the ribose, but is not limited thereto. Exemplary ribose includes, but is not limited to, 2 '-( C1-C6) alkoxy ribose, 2 '-( C5- C14) aryloxy ribose, 2 ', 3 ' -didehydro- ribose, Deoxy-3'-fluoro-ribose, 2'-deoxy-3'-chlorobenzene, 2'-deoxy-3'-amino ribose, (C5-C14) aryloxy ribose, ribose, 2'-deoxyribose, 2'-deoxy-3'- , 3'-dideoxyribose, 2'-haloribose, 2'-fluororibose, 2'-chlororibose and 2'-alkylribose such as 2'- Linked and other " locked "or" LNA ", bicyclic sugars, such as the anomeric nucleotides, the 1'-a-anomer nucleotides, the 2'- But are not limited to, bicyclic sugar modifications (see, for example, PCT Published Applications WO 98/22489, WO 98/39352 and WO 99/14226; Article refers to 6.26849 million) and (No. 6,794,499).

The additive material is a compound added to the composition that alters the stability, activity, and / or duration of one or more of the ingredients in the composition. In certain embodiments, the composition is an amplification reaction composition. In certain embodiments, the additive material deactivates the contaminating enzyme, stabilizes protein folding, and / or reduces aggregation. Exemplary additives that may be included in the amplification reaction include, but are not limited to, bine, formamide, KCl, CaCl ₂ , MgOAc, MgCl ₂ , NaCl, NH ₄ OAc, NaI, Na (CO ₃ ) ₂ , LiCl, MnOAc, , Trehalose, demethylsulfoxide ("DMSO"), glycerol, ethylene glycol, dithiothreitol ("DTT"), ( Thermoplasma pyrophosphatase, bovine serum albumin ("BSA"), propylene glycol, glycine amide (including but not limited to acidophilum ) inorganic pyrophosphatase glycinamide, CHES, Percoll ^TM , aurintricarboxylic acid, Tween 20, Tween 21, Tween 40, Twin 60, Twin 85, Brij 30, NP-40, Triton X-100, CHAPS, CHAPSO N-dimethylamine-N-oxide), Zwittergent 3-10, , Escherichia coli SSB, RecA, nicking endonuclease, 7-deaza G, dUTP, UNG, anionic detergent, cationic detergent, nonionic detergent, zwittergent, sterol, osmolyte, cation, and any other chemical, protein or cofactor capable of altering the amplification efficiency. In certain embodiments, more than one additive material is included in the amplification reaction. According to the present invention, the additive materials may be added for the purpose of improving the selectivity of the primer anneal, if they do not interfere with the activity of RNase H.

The term "thermostable" as used herein, when applied to an enzyme, refers to the ability to maintain its biological activity at elevated temperature conditions (e.g., 55 캜 or higher) Refers to an enzyme that retains its biological activity later on. Thermostable polynucleotide polymerases are particularly useful in PCR amplification reactions.

As used herein, "amplifying polymerase activity" refers to the enzyme activity that catalyzes the polymerization of deoxyribonucleotides. Generally, the enzyme will initiate synthesis at the 3'-end of the primer annealed to the nucleic acid template sequence and will proceed in the 5'-terminal direction of the template strand. In certain embodiments, "amplified polymerase activity" is a thermostable DNA polymerase.

As used herein, thermostable polymerases are thermally stable enzymes, eliminating the need to add enzymes before each PCR cycle.

Non-limiting examples of thermostable DNA polymerases include thermostable bacteria Thermus aquaticus aquaticus) (Taq polymerase), Thermococcus sseomeoseu pillar's (Thermus thermophilus) (Tth polymerase), Thermococcus litoralis (Thermococcus litoralis (Tli or VENT ^TM polymerase), Pyrococcus furiosus), (Pfu or DEEPVENT ^TM. polymerase), Rhodococcus woosiyi (Pyrococcus Pyro and Bacillus stearothermophilus (Bst polymerase), Sulfolobus acidocaldarius (Sac polymerase), Thermoplasma aciduria ( Bacillus stearothermophilus), Bacillus stearothermophilus Thermoplasma acidophilum (Tac polymerase), Thermus rubber ( Thermus rubber (Tru polymerase), Thermus brockianus ) (DYNAZYME ^TM Polymerase i (Tne Polymerase), Thermotoga ( Thermotoga) but are not limited to, maritime (Tma) and other species of the Thermotoga genus (Tsp polymerase), and Methanobacterium thermoautotrophicum (Mth polymerase) It does not. The PCR reaction may include one or more thermostable polymerase enzymes having complementary properties that cause more efficient amplification of the target sequence. For example, a nucleotide polymerase having a high degree of processivity (ability to replicate a large nucleotide fragment) can be used in combination with another nucleotide polymer having the ability to proofread (ability to correct errors during extension of the target nucleic acid sequence) By complementing with Razer, a PCR reaction can be produced that can replicate long target sequences with high fidelity. The thermostable polymerase can be used in its wild-type form. Alternatively, to facilitate PCR reactions, the polymerase can be modified to include a fragment of the enzyme, or to include mutations that provide beneficial properties. In one embodiment, the thermostable polymerase may be a Taq polymerase. And a plurality of Taq polymerase variants with improved properties are known, AmpliTaq ^TM, AmpliTaq ^TM, testosterone pel (Stoffel) fragment, SuperTaq ^TM , SuperTaq ^TM plus, LA Taq ^TM , LApro Taq ^TM and EX Taq ^TM , but are not limited thereto. In yet another embodiment, the thermostable polymerase used in the multiplex amplification reaction of the present invention is an AmpliTaq Stapel fragment.

RNA target  The reverse transcriptase of the nucleic acid sequence - PCR  Amplification

One of the most widely used techniques for gene expression studies uses first-strand cDNA for the mRNA sequence (s) as a template for amplification by PCR.

The terms " reverse transcriptase activity "and" reverse transcription "refer to the use of RNA strands as templates to produce RNA-dependent DNA polymer Refers to the enzyme activity of a polymerase class having the characteristics of lysine.

The term " reverse transcriptase-PCR "or" RNA PCR "refers to DNA-dependent DNA polymerase prior to multiple cycles of primer extension. RNA polymerase or reverse transcriptase, or reverse transcriptase, Is a PCR reaction using an enzyme having an activity. Multiplex PCR typically refers to PCR reactions that involve more than one primer in a single reaction, resulting in more than one type of amplification product in a single reaction.

Exemplary reverse transcriptases include mutant forms of M-MLV-RT disclosed in U.S. Patent No. 4,943,531, Moloney murine leukemia virus (M-MLV) RT, U.S. Patent No. 5,405,776, Including the bovine leukemia virus (RTV) RT, the Rous sarcoma virus (RTV) RT, the Avian Myeloblastosis Virus (RTV) RT and the reverse transcriptase enzymes disclosed in U.S. Patent No. 7,883,871 But is not limited thereto.

The reverse transcriptase-PCR process, performed by end-point or real-time analysis, involves two distinct molecular syntheses: (i) synthesis of cDNA from RNA templates; And (ii) replication of the newly synthesized cDNA via PCR amplification. In an attempt to address the technical problems often associated with reverse transcriptase-PCR, a number of protocols have been developed that take into account three basic steps of the process: (a) denaturation of RNA and hybridization of reverse primers; (b) synthesis of cDNA; And (c) PCR amplification. In a so-called " uncoupled "reverse transcriptase-PCR procedure (e.g., a second-stage reverse transcriptase-PCR), reverse transcription is performed as an independent step using optimal buffer conditions for reverse transcriptase activity. Following cDNA synthesis, the reaction is diluted to reduce the concentration of MgCl ₂ and deoxyribonucleotide triphosphate (dNTP) under conditions optimal for Taq DNA polymerase activity and PCR is performed according to standard conditions (US Pat. No. 4,683,195 No. 4,683,202). Conversely, the "coupled" RT PCR method uses conventional buffers optimized for reverse transcriptase and Taq DNA polymerase activity. In one embodiment, the annealing of the reverse primer is an independent step performed prior to the addition of the enzyme, which is then added to a single reaction vessel. In another embodiment, the reverse transcriptase activity is a component of a thermostable Tth DNA polymerase. Annealing and cDNA synthesis are performed in the presence of Mn ^{2 +} , and then PCR is performed in the presence of Mg ^{2 +} after removal of Mn ^{2 +} by a chelating agent. Finally, a "continuous" method (e. G., A first-step reverse transcriptase-PCR) combines the three reverse transcriptase-PCR steps into one continuous reaction to open a reaction vessel for the addition of a constituent or enzyme . Continuous reverse transcriptase-PCR is a two enzyme system using a single enzyme system using reverse transcriptase activity of thermostable Taq DNA polymerase and Tth polymerase, and an AMV RT and Taq DNA polymerase at an initial temperature of 65 ° C Has come. The RNA denaturation step may be omitted.

In certain embodiments, one or more primers can be labeled. The term "label", "detectable label", or "marker" or "detectable marker", used interchangeably herein, refers to a nucleotide, a nucleotide polymer, Or any chemical moiety attached to a nucleic acid binding factor, which attachment may be by covalent or non-covalent attachment. Preferably, the label is detectable and allows the practitioner of the present invention to detect the nucleotide or nucleotide polymer. Detectable labels include luminescent molecules, chemiluminescent molecules, fluorescent dyes, colored molecules, radioactive isotopes or scintillants. Detectable label is further (biotin, avidin, streptavidin, HRP, protein A (protein A), protein G, an antibody or a fragment thereof, Grb2, polyhistidine, Ni ^{2 +,} such as the FLAG tag, myc tag) any Acceptor / acceptor, acridinium ester, pigments and calorimetric substrates, as well as useful linker molecules, heavy metals, enzymes (including alkaline phosphatases, peroxidases and luciferases) . Also, as in the case of surface plasmon resonance, a change in mass is expected to be considered as a detectable marker. Skilled artisans will readily recognize useful detectable labels not mentioned above that may be used in the practice of the present invention.

The first-step reverse transcriptase-PCR provides several advantages over the unconjugated reverse transcriptase-PCR. The first-step reverse transcriptase-PCR requires less handling of the reaction mixture reagents and nucleic acid products than the unconjugated reverse transcriptase-PCR (for example, between reaction steps, , Thus less labor intensive and reduces the required number of person hours. The first-stage reverse transcriptase-PCR also requires less sample and reduces the risk of contamination. The sensitivity and specificity of the first stage reverse transcriptase-PCR has been shown to be well suited for the study of the expression levels of one or several genes in a given sample, or for the detection of pathogenic RNA. Traditionally, this process has been limited to the use of gene-specific primers to initiate cDNA synthesis.

The ability to measure the kinetics of PCR reactions by on-line detection combined with this reverse transcriptase-PCR technique enabled accurate and precise quantification of the number of RNA copies with high sensitivity. This can be accomplished using fluorescent double-labeled hybridization probes, such as the 5 'fluorogenic nuclease assay ("TaqMan ^™ ") or the endonuclease assay ("CataCleave ^™ " PCR products by means of dual-labeled hybridization probe technology, fluorescence monitoring during the amplification process and measurement of PCR products.

CataCleave ^TM The probe  Real time PCR

Detection of amplicon after amplification is difficult and takes a long time. A real-time method has been developed to monitor amplification during the PCR process. These methods generally use fluorescently labeled probes that bind to newly synthesized DNA, or pigments that increase fluorescence emission when inserted between double-stranded DNA. Real-time detection methods are applicable to PCR detection of SNPs in genomic DNA or genomic RNA.

The probe is generally designed such that donor emission in the absence of the target is quenched by fluorescence resonance energy transfer (FRET) between the two chromophores. The donor chromophore can transfer energy to the acceptor chromophore in the excited state when it is close to the acceptor chromophore. Such transitions are always non-radioactive and occur through dipole-dipole coupling. Any process that sufficiently increases the distance between the two chromophores will reduce the FRET efficiency and allow donor chromophore release to be detected radiatively. Typical donor chromophores include FAM, TAMRA, VIC, JOE, Cy3, Cy5, and Texas Red. The acceptor chromophore is chosen such that its excitation spectrum overlaps the donor emission spectrum. An example of such a pair is FAM-TAMRA. There are also non-fluorescent acceptors that can quench various donors. Other suitable examples of donor-acceptor FRET pairs will be known to those skilled in the art.

Examples of conventional FRET probes that can be used for real-time detection of PCR include molecular beacons (e.g., U.S. Patent No. 5,925,517), TaqMan ^™ probes (see, for example, U.S. Patent No. 5,210,015 and 5,487,972), and CataCleave ^TM probes (e.g., U.S. Patent No. 5,763,181). Wherein the molecular beacon is a single strand oligonucleotide in which the probe is designed to form a secondary structure in which the donor and acceptor chromophore are at a close distance and the donor emission is reduced in the unbonded state. At the appropriate reaction temperature, the beacon is unfolded and binds specifically to the ampicillin. After spreading, the distance between donor and acceptor chromophore is increased to reverse the FRET, and special device can be used to monitor donor emission. TaqMan ^TM and CataCleave ^TM techniques differ from molecular beacons in that the FRET probe used is cleaved so that the donor and acceptor chromophore are sufficiently separated to invert the FRET.

The TaqMan ^TM technology uses a single stranded oligonucleotide probe in which the 5 'terminus is labeled with donor chromophore and the 3' terminus is labeled with acceptor chromophore. The DNA polymerase used for amplification should have 5 '→ 3' exonuclease activity. The TaqMan ^™ probe binds to one strand of the amplicon at the same time the primer binds. DNA polymerase I, while elongating the primer, the polymerase will run into the TaqMan ^TM probes that are coupled in the end. At this point, the exonuclease activity of the polymerase will degrade the TaqMan ^™ probe sequentially from the 5 'end. Once the probe is degraded, the mononucleotide containing the probe is released into the reaction buffer. The donor moves away from the prisoner, and the FRET is reversed. Monitor release from the donor to confirm probe cutting. Because of the way in which TaqMan ^TM works, a particular amplicon can be detected only once per cycle of the PCR. TaqMan ^TM Elongation of the primers with the target area will prevent further binding of TaqMan ^TM probe until to produce a double-stranded product, the amplicon is to be modified in the subsequent PCR cycles.

U.S. Patent No. 5,763,181, whose contents are incorporated herein by reference, discloses another real-time detection method (referred to as "CataCleave ^TM "). CataCleave ^TM Technology is distinguished from TaqMan ^TM in that cleavage of the probe is achieved by a second enzyme that does not have polymerase activity. The CataCleave ^TM probe has an intramolecular sequence that is the target of an endonuclease, such as, for example, a restriction enzyme or RNase. In one example, the CataCleave ^TM probe has a chimeric structure in which the 5 'and 3' ends of the probe are composed of DNA and the cleavage site is RNA. The DNA sequence portion of the probe is labeled at both ends or internally with a FRET pair. The PCR reaction involves the RNase H enzyme, which specifically cleaves the RNA sequence portion of the RNA-DNA double strand. After cleavage, the two halves of the probe dissociate from the target ampicrone at the reaction temperature and diffuse into the reaction buffer. As the donor and recipient are separated, the FRET can be inverted in the same manner as the TaqMan ^™ probe and monitored for donor release. Cleavage and dissociation regenerate sites for additional CataCleave ^™ binding. In this way, a single amplicon can be used as a target until the primer is extended through the CataCleave ( ^TM) probe binding site, or for multiple-time probe cleavage.

CataCleave ^TM Of the probe  Labeling

The term "probe" encompasses polynucleotides that comprise a specific nucleic acid sequence, for example, a specific portion designed to sequence-specifically hybridize to a complementary region of a target nucleic acid sequence. In one embodiment, the oligonucleotide probe has a length of 15 to 60 nucleotides. More preferably, the oligonucleotide probe has a length of 18 to 30 nucleotides. The specific sequence and length of the oligonucleotide probe of the present invention depends in part on the nature of the target polynucleotide to which it binds. The bonding position and length can be varied to achieve the appropriate annealing and melting characteristics in particular embodiments. The guidelines for such design choices are described in numerous references, such as TaqMan ^TM analysis or CataCleave ^TM , as disclosed in U.S. Patent Nos. 5,763,181, 6,787,304, and 7,112,422, the contents of which are incorporated herein by reference. Can be found in references.

In certain embodiments, the probe is "substantially complementary" to the target nucleic acid sequence.

As used herein, the term " substantially complementary "refers to both nucleic acid strands sufficiently complementary in sequence to be able to anneal to form stable double strands. Complementarity need not be perfect; For example, between any two nucleic acids, any number of unmatched base pairs may be present. However, if the number of mismatches is so large that the hybridization is not achieved even under the least stringent hybridization conditions, the sequence is not a substantially complementary sequence. In the present specification, when two sequences are referred to as " substantially complementary ", this means that the sequences are sufficiently complementary to each other to hybridize under the selected reaction conditions. The relationship between nucleic acid complementarity and stringency of sufficient hybridization to achieve specificity is well known in the art. Substantially complementary two strands may, for example, be either fully complementary, or hybridized under conditions where one hybridization condition is sufficient, for example, as long as it is sufficient to enable discrimination between a pairing sequence and a non-pairing sequence Or a plurality of mismatches. Thus, a "substantially complementary" sequence refers to a sequence having, in the double-stranded region, any base pair complementarity of 100, 95, 90, 80, 75, 70, 60, 50% .

As used herein, "selected region" refers to the polynucleotide sequence of the target DNA or cDNA annealing to the RNA sequence of the probe. In one embodiment, the "selected region" of the target DNA or cDNA is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length.

As used herein, site-specific RNase H cleavage is the cleavage of an RNA moiety in a Catacleave ^™ probe, which is entirely complementary to the target DNA sequence and hybridizes therewith to form an RNA: DNA heterodimer double strand Quot;

The Catacleave ^TM If the RNA moiety in the probe comprises a single nucleotide polymorphism and the target DNA sequence comprises a wild-type sequence at the position of the polymorphism, the formation of an RNA: DNA heterozygous double strand between the Catacleave ^TM probe and the wild- Resulting in a single nucleotide mismatch, which prevents cleavage of the RNA moiety in the Catacleave ^™ probe by RNase H.

Likewise, if the target DNA sequence comprises a SNP sequence and Catacleave ^TM If the RNA moiety of the probe comprises a wild-type sequence at a polymorphic position, Catacleave ^TM RNA between the wild-type target DNA sequence comprising the probe and the SNP sequence: and results in a single nucleotide mismatch is formed at the polymorphic position of the double-stranded DNA releasing, which Catacleave by RNase H ^TM Prevents cleavage of the RNA moiety in the probe.

As used herein, Catacleave ^TM The term "label" or "detectable label" of a probe refers to any label comprising a fluorochrome compound attached to the probe by either covalent or non-covalent means.

As used herein, "fluorochrome" refers to a fluorescent compound that is excited by light of a shorter wavelength than that emitted and then emits light. The term " fluorescent donor or fluorescent donor "refers to a fluorescent dye that emits light as measured in the assay described herein. More particularly, the fluorescent donor provides light that is absorbed by the fluorescent receptor. The term " fluorescent acceptor "refers to a second fluorescent dye or quencher molecule that absorbs light emitted from a fluorescent donor. The second fluorescent dye absorbs light emitted from the fluorescent donor and emits light of longer wavelength than the light emitted from the fluorescent donor. The quencer molecule absorbs light emitted from the fluorescent donor.

^{Alexa Fluor TM 350, Alexa Fluor TM} 430, Alexa Fluor TM 488, Alexa Fluor TM 532, Alexa Fluor TM 546, Alexa Fluor TM 568, Alexa Fluor TM 594, Alexa Fluor TM 633, Alexa Fluor TM 647, Alexa Fluor TM 660, Alexa Fluor ^TM 680, 7-diethylaminocoumarin-3-carboxylic acid, Fluorescein, Oregon Green 488, Oregon Green 514, Tetramethylrhodamine, Rhodamine X, Texas Red Red dye), QSY 7, QSY33, Dabcyl, BODIPY FL, BODIPY 630/650, BODIPY 6501665, BODIPY TMR-X, BODIPY TR-X , dialkylamino coumarin, Cy5.5, Cy5, Cy3.5, Cy3 , DTPA (Eu 3 +) -AMCA and TTHA (Eu ^{+ 3),} any of the light-emitting molecules, including AMCA, Preferably a fluorescent dye and / or a fluorescent quencher may be used in the practice of the present invention.

In one embodiment, the 3 ' terminal nucleotide of the oligonucleotide probe is blocked or treated to be unable to elongate by the nucleic acid polymerase. Such blocking is conveniently accomplished by attaching a reporter or a quencher molecule to the 3 ' position of the probe.

In one embodiment, the reporter molecule is a fluorescent organic dye that is induced to attach to the terminal 3 'or terminal 5' of the probe by a linking moiety. Preferably, the quencer molecules are also organic or fluorescent in nature, according to embodiments of the present invention. For example, in a preferred embodiment of the present invention, the quencer molecule is fluorescent. Generally, regardless of whether the quencer molecule is emitting fluorescence or simply the energy transferred from the reporter by non-radiative decay, the absorption band of the quencer substantially overlaps with the fluorescent emission band of the reporter molecule . A non-fluorescent quencher molecule that absorbs energy from an excited reporter molecule, but does not radially emit energy, is referred to in this application as a chromogenic molecule.

An exemplary reporter-quencher pair can be selected from xanthene and rhodamine pigments, including fluorescein. A number of suitable forms of these compounds having substituents on their phenyl moieties that can be used as binding sites or binding functionalities for attachment to oligonucleotides are widely available. Another group of fluorescent compounds is naphthylamine, which has an amino group at the alpha or beta position. Examples of such naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalenesulfonate and 2-p-toidinyl-6-naphthalenesulfonate . Other dyes include acridine, N- (p- (2-benzyisazolyl) aminocarbonyl, such as 3-phenyl-7-isocyanatocoumarin, 9-isothiocyanatoacridine and acridine orange, ) Phenyl) maleimide, benzoxadiazole, stilbene, pyrene, and the like.

In one embodiment, the reporter and quencher molecules are selected from fluorocaine and rhodamine pigments.

There are a number of linking moieties and methods for attaching the reporter or quencer molecules to the 5 ' or 3 ' end of an oligonucleotide, exemplified by the following references: Eckstein Ed., Oligonucleotides and Analogues: A Practical Approach Press, Oxford, 1991); Zuckerman et al., Nucleic Acids Research, 15: 5305-5321 (1987) (3 'thiol group on oligonucleotides); Sharma et al., Nucleic Acids Research, 19: 3019 (1991) (3 'sulfhydryl); (5 ') by Aminolink.TM. II, available from Giusti et al., PCR Methods and Applications, 2: 223-227 (1993) and Fung et al., U.S. Patent No. 4,757,141 (Applied Biosystems, Foster City, Calif. Phosphoamino group), US 4,739,044 (3 'aminoalkylphosphoryl group) by Stabinsky; Agrawal et al., Tetrahedron Letters, 31: 1543-1546 (1990) (attachment by phosphoramidate linkages); Sproat et al., Nucleic Acids Research, 15: 4837 (1987) (5 'mercapto group); Nelson et al., Nucleic Acids Research, 17: 7187-7194 (1989) (3 'amino group), and the like.

Rhodamine and fluorocaine dyes may also conveniently be prepared by solid phase synthesis with colorant derivatives including phosphoramidite moieties, such as those described in Woo et al., U.S. Patent Nos. 5,231,191; And in the last step of U.S. Patent No. 4,997,928 by Hobbs, Jr., attached to the 5'hydroxy group of the oligonucleotide.

As a solid support CataCleave ^TM Of the probe  Attach

In one embodiment, the oligonucleotide probe can be attached to a solid support. Different probes may be attached to the solid support and used for simultaneous detection of different target sequences present in the sample. Reporter molecules with different fluorescence wavelengths can be used in the different probes, thereby enabling hybridization with different probes to be independently detected.

Examples of suitable solid support species for immobilization of oligonucleotide probes include controlled porous glass, glass plates, polystyrene, avidin coated polystyrene bead cellulose, nylon, acrylamide gel and activated dextran, controlled porous glass pore glass, CPG), glass plates and high cross-linked polystyrenes. These solid supports are preferred for hybridization and diagnostic studies due to their chemical stability, ease of functionalization, and well defined surface area. Solid supports such as controlled porous glass (500 ANGSTROM, 1000 ANGSTROM) and non-swelling, highly crosslinked polystyrene (1000 ANGSTROM) are particularly preferred in view of their compatibility with oligonucleotide synthesis.

Oligonucleotide probes can be attached to solid supports in a variety of ways. For example, a probe can be attached to a solid support by attachment of the 3 ' or 5 ' terminal nucleotide of the probe to the solid support. However, the probe may be attached to the solid support by a linker that keeps the probe away from the solid support. The linker preferably has a length of at least 30 atoms, more preferably at least 50 atoms.

Hybridization of a probe immobilized on a solid support generally requires that the probe be separated from the solid support by at least 30 atoms, more preferably by at least 50 atoms. To achieve this separation, the linker preferably comprises a spacer disposed between the linker and the 3 'nucleoside. For oligonucleotide synthesis, to dissociate the oligonucleotide from the solid support, the linker arm is attached to the 3'-OH of the 3 'nucleoside by an ester linkage that can be cleaved with a basic reagent.

A variety of linkers are known in the art that are used to attach oligonucleotides to solid supports. The linker may be composed of any compound that does not significantly interfere with the hybridization of the probe with the target sequence attached to the solid support. Linkers can be composed of homopolymeric oligonucleotides that can be easily added to the linker by automated synthesis. Alternatively, polymers such as functionalized polyethylene glycols can be used as linkers. Such polymers are preferred over homopolymeric oligonucleotides because they do not significantly interfere with the hybridization of the probe and the target oligonucleotide. Polyethylene glycol is particularly preferred because it is commercially available, soluble in both organic and aqueous media, easy to functionalize, and completely stable in oligonucleotide synthesis and post-synthesis conditions.

The bond between the solid support, the linker and the probe is preferably not cleaved during removal of the base protecting group under high temperature, basic conditions. Examples of preferred linkages include carbamate and amide linkages. Immobilization of probes is well known in the art, and one skilled in the art can determine the immobilization conditions.

According to one embodiment of the method, the CataCleave ^TM probe is immobilized on a solid support. Wherein the CataCleave ^TM probe comprises a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected portion of a target DNA sequence comprising a polymorphism, Is substantially complementary to the DNA sequence adjacent to the selected region of the DNA sequence. The probe is then contacted with a nucleic acid sample in the presence of RNase H, under conditions which allow the RNA sequence in the probe to form a complementary DNA sequence and a RNA: DNA heterodimer double strand in a PCR fragment comprising the polymorphism. RNase H cleavage of the RNA: DNA heterozygous double stranded RNA sequence results in a real-time increase in the signal emitted from the label on the probe, and the increase in signal indicates the presence of the polymorphism in the target DNA.

According to another embodiment of the method, a CataCleave ^TM probe immobilized on a solid support comprises a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA ogrtks sequence of the probe comprises a wild-type DNA sequence at a polymorphic position The DNA nucleotide sequence of the probe is substantially complementary to the DNA sequence adjacent to the selected region of the target DNA sequence. The probe is then contacted with a nucleic acid sample in the presence of RNase H, under conditions which allow the RNA sequence in the probe to form a complementary DNA sequence and a RNA: DNA heterodimer double strand in a PCR fragment comprising the polymorphism. If the target DNA sequence comprises a polymorphism, a mismatch at the polymorphic position in the RNA: DNA duplex inhibits RNase H cleavage of the RNA sequence in the RNA: DNA duplex double strand, which results in the release Resulting in a real-time reduction of the signal, wherein the reduction of the signal indicates the presence of polymorphism in the target DNA.

Immobilization of the probe to the solid support allows the target sequence hybridized to the probe to be easily separated from the sample. In a later step, the separated target sequence can be separated from the solid support and processed (e.g., purified, amplified) according to the specific needs of the researcher according to methods well known in the art.

Catacleave ^TM Of the probe RNase  H cutting

RNase H hydrolyzes RNA in RNA-DNA hybrids. RNase H, first identified in calf thymus, was subsequently found in a variety of organisms. Indeed, RNase H activity is universally found in eukaryotes and bacteria. Although RNase H constitutes a family of proteins with varying molecular weights and nucleolytic activities, substrate requirements are similar for a variety of isotypes. For example, most of the RNase Hs studied so far function as endo-nuclease and have a divalent cation (for example, Mg ^{2 +} , ^< RTI ID = 0.0 & ^{gt ;} , Mn < ^{2 +} & gt ^; ).

In prokaryotes, RNase H has been cloned and widely characterized (Crooke et al., (1995) Biochem J, 312 (Pt 2), 599-608; Lima et al., (1997) J Biol Chem, 272, 27513- Lim, et al. (2007) Mol Pharmacol, 71, 83-91; Lima et al., (1997) Biochemistry, 36, 390-398; Lima et al., (1997) J Biol Chem, 272, 18191-18199; (2007) Mol Pharmacol, 71, 73-82; Lima et al., (2003) J Biol Chem, 278, 14906-14912; Lima et al., (2003) J Biol Chem, 278, 49860-49867; Natl. Acad. Sci. USA, 1990, 87, 8587-8591). For example, E. coli RNase HII has a length of 213 amino acids and RNase HI has a length of 155 amino acids. E. coli RNase HII has only 17% homology with E. coli RNase HI. S, RNase H cloned from S. typhimurium differed only 11 positions from E. coli RNase HI and had a length of 155 amino acids (Itaya, M. and Kondo K., Nucleic Acids Res., 1991, 19, 4443-4449).

Proteins exhibiting RNase H activity were also cloned and purified from a number of viruses, other bacteria and yeast (Wintersberger, U. Pharmac. Ther., 1990, 48, 259-280). In many cases, proteins with RNase H activity have been shown to be fusion proteins in which RNase H is fused to another enzyme, often an amino or carboxy terminus of a DNA or RNA polymerase. Although the RNase H domain has been consistently shown to have a high degree of homology with E. coli RNase HI, the molecular weight and other properties of the fusion protein vary widely because the different domains are quite diverse.

Two types of RNase H have been defined in high eukaryotes based on differences in molecular weight, the effect of divalent cations, sulfhydryl agents and immunological cross-reactivity (Busen et al., Eur. J. Biochem. 1977, 74, 203-208). The RNase HI enzyme has a molecular weight in the range of 68-90 kDa, is activated by Mn ^{2 +} or Mg ^{2 +} , and is reported to be insensitive to sulfhydryl agents. In contrast, RNase HII enzyme was reported to have a molecular weight of 31-45 kDa range, require a Mg ^{2 +,} and very sensitive to the sulfinyl drill agonists and inhibited by Mn ^{2 +} (Busen, W., and 1982, 257, < RTI ID = 0.0 > J. < / RTI > Biol. Chem., 1975, 52, 179-190; Kane, CM, Biochemistry, 1988,27, 3187-3196; 7106-7108).

Enzymes with RNase HII properties have also been purified from the human placenta to close homologous (Frank et al., Nucleic Acids Res., 1994, 22, 5247-5254). This protein has a molecular weight of about 33 kDa and is active at a pH in the range of 6.5-10, with an optimum pH of 8.5-9. The enzyme requires a Mg ^{+ 2,} is inhibited by Mn ^{2 +} and n- ethyl maleimide. The products of the cleavage reaction have 3 ' hydroxy and 5 ' phosphate ends.

Detailed comparisons of RNases isolated from various species are available from Ohtani N, Haruki M, Morikawa M, Kanaya S. J Biosci Bioeng. 1999; 88 (1): 12-9.

Examples of RNase H enzymes that can be used in this embodiment include pyrococcus furiosus , Pyrococcus horikoshi ), Thermococcus thermostable RNase H enzymes isolated from thermophilic organisms such as, for example, litoralis or Thermus thermophilus .

Other RNase H enzymes that may be used in this embodiment are disclosed, for example, in U.S. Patent No. 7,422,888 by Uemori or U.S. Patent Application Publication No. 2009/0325169 by Walder, the contents of which are incorporated herein by reference Are incorporated herein.

In one embodiment, the RNase H enzyme has an amino acid sequence that is 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to the amino acid sequence of Pfu RNase HII (SEQ ID NO: Homozygous, heat stable RNase H.

MKIGGIDEAG RGPAIGPLVV ATVVVDEKNI EKLRNIGVKD SKQLTPHERK NLFSQITSIA 60

DDYKIVIVSP EEIDNRSGTM NELEVEKFAL ALNSLQIKPA LIYADAADVD ANRFASLIER 120

RLNYKAKIIA EHKADAKYPV VSAASILAKV VRDEEIEKLK KQYGDFGSGY PSDPKTKKWL 180

EEYYKKHNSF PPIVRRTWET VRKIEESIKA KKSQLTLDKF FKKP (SEQ ID NO: 13)

Homology can be obtained, for example, by the computer program DNASIS-Mac (Takara Shuzo), the computer algorithm FASTA (version 3.0; Pearson, WR et al., Pro.Natl.Acad. Sci., 85: 2444-2448, 1988) version 2.0, Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997).

In another embodiment, the RNase H enzyme has at least one homology region 1-4, corresponding to positions 5-20, 33-44, 132-150, and 158-173 of SEQ ID NO: 13, Thermal stability is RNase H. This region of homology is called Pyrococcus furiosis , Pyrococcus horikoshi), Thermo Lactococcus coder Karen sheath (Thermococcus kodakarensis), Douce (Archeoglobus booth Pro extracted with ahreuke Ogle profundus), solve booth to display ahreuke Ogle (Archeoglobus fulgidis , Thermococcus celer , and Thermococcus litoralis ) RNase HII polypeptide sequence (see Figure 10).

Homology region 1: GIDEAG RGPAIGPLVV (SEQ ID NO: 20, corresponding to positions 5-20 of SEQ ID NO: 13)

Homology region 2: LRNIGVKD SKQL (SEQ ID NO: 21, corresponding to position 33-44 of SEQ ID NO: 13)

Homology region 3: HKADAKYPV VSAASILAKV (SEQ ID NO: 22, corresponding to position 132-150 of SEQ ID NO: 13)

Homology region 4: KLK KQYGDFGSGY PSD (SEQ ID NO: 23, corresponding to position 158-173 of SEQ ID NO: 13)

In one embodiment, the RNase H enzyme is 50%, 60% or more homologous to the polypeptide sequence of SEQ ID NO: 20, 21, 22 or 23. Is a thermostable RNase H, comprising at least one of homology regions having a sequence identity of 70%, 80%, 90%.

In yet another embodiment, the RNase H enzyme is at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% identical to the amino acid sequence of the Thermus thermophilus RNase HI / RTI > is a thermostable RNase H having a < RTI ID = 0.0 >

MNPSPRKRVA LFTDGACLGN PGPGGWAALL RFHAHEKLLS GGEACTTNNR MELKAAIEGL

KALKEPCEVD LYTDSHYLKK AFTEGWLEGW RKRGWRTAEG KPVKNRDLWE ALLLAMAPHR

VRFHFVKGHT GHPENERVDR EARRQAQSQA KTPCPPRAPT LFHEEA (SEQ ID NO: 25)

In another embodiment, the RNase H enzyme is a thermostable RNase H comprising at least one of homologous sites 5 to 8 corresponding to positions 23-48, 62-69, 117-121 and 141-152 of SEQ ID NO: to be. These homology regions are defined by sequence placement.

Haemophilus influenzae influenzae ), Sseomeoseu Thermo Phillies (Thermus thermophilis), sseomeoseu aqua tee Syracuse (Thermus acquaticus , Salmonella enterica enterica ) and Agrobacterium tumefaciens RNase HI polypeptide sequences (see FIG. 11).

Homology region 5: K * V * LFTDG * C * GNPG * GG * ALLRY (SEQ ID NO: 29, corresponding to positions 23-48 of SEQ ID NO: 25)

Homology region 6: TTNNRMEL (SEQ ID NO: 30, corresponding to position 62-69 of SEQ ID NO: 25)

Homology region 7: KPVKN (SEQ ID NO: 31, corresponding to position 117-121 of SEQ ID NO: 25)

Homology region 8: FVKGH * GH * ENE (SEQ ID NO: 32, corresponding to position 141-152 of SEQ ID NO: 25)

In yet another embodiment, the RNase H enzyme is selected from the group consisting of 50%, 60%, 30%, 30%, 30%, or 32% And 70%, 80%, 90% sequence homology with at least one of the homologous sites 4 to 8.

As used herein, "sequence identity" refers to a range of sequences that are identical, or functionally or structurally similar, based on amino acid to amino acid over a comparison range. Thus, a "percentage of sequence identity" can be determined, for example, by comparing two sequences that are optimally aligned over a comparison range, determining the number of positions where the same amino acid exists in both sequences, Dividing the number of matched positions by the total number of positions in the comparison range (i. E., Range size), and multiplying the result by 100 to yield a percentage of sequence identity. ≪ RTI ID = 0.0 > have.

In certain embodiments, RNase H can be modified to produce a hot start " inducible "RNase H.

As used herein, the term " modified RNase H "may be RNase H, which inversely couples or reversely binds to an inhibitory factor resulting in the loss of endonuclease activity of RNase H . The release or decoupling of the inhibitor from the RNase H completely or at least partially restores the endonuclease activity of the RNase H. Approximately 30 to 100% of the endonuclease activity of pure RNase H may suffice. The inhibitory factor may be a ligand or a chemical variant. The ligand may be an antibody, an aptamer, a receptor, a cofactor, or a chelating agent. The ligand may bind to the active site of the RNase H enzyme, thereby inhibiting the enzyme activity or binding to a site remote from the active site of the RNase H enzyme. In some embodiments, the ligand can induce a structural change. The chemical modification may be (for example, by formaldehyde) crosslinking or acylation. Release or decoupling of the inhibitor from RNase H can be accomplished by heating the sample or coupled RNase H (inert) to a temperature of from about 65 [deg.] C to about 95 [deg.] C or higher and / Can be achieved by a lowering step.

As used herein, the hot start " inducible "RNase H activity refers to the modified RNase H described herein, which has an endo-nuclease enzyme activity that can be modulated by its association with the ligand. In the relaxed condition, the RNase H endo-nuclease enzyme activity is activated, whereas under non-permissive conditions, the enzyme activity is inhibited. In some embodiments, the enzymatic activity of the modified RNase H can be inhibited at a temperature at which reverse transcription can be performed, i.e. at about 42 ° C, and at elevated temperatures observed in the PCR reaction, ie, at about 65 ° C to 95 ° C, . Modified RNase H with these properties is referred to as being "heat inducible ".

 In another embodiment, the enzymatic activity of the modified RNase H can be modulated by changing the pH of the solution containing the enzyme.

As used herein, a "hot start" enzyme composition is inhibited at a temperature at which replication is not allowed, i.e., from about 25 ° C to about 45 ° C, and is at a temperature suitable for the PCR reaction, It is activated at 95 ℃. In certain embodiments, the "hot start" enzyme composition may have a 'hot start' RNase H and / or a 'hot start' thermostable DNA polymerase known in the art.

Crosslinking of the RNase H enzyme can be carried out, for example, using formaldehyde. In one embodiment, the thermostable RNase H can undergo controlled and limited crosslinking using formaldehyde. By heating the amplification reaction composition, including the active modified RNase H, to about 95 ° C or higher for extended periods of time, for example about 15 minutes, the cross-linking is reversed and the RNase H activity is restored.

Generally, the lower the degree of crosslinking, the higher the endonuclease activity of the enzyme after cross-linking reversal. The degree of crosslinking can be controlled by varying the concentration of formaldehyde and the duration of the crosslinking reaction. For example, about 0.2% (w / v), about 0.4% (w / v), about 0.6% (w / v), or about 0.8% (w / v) formaldehyde for crosslinking the RNase H enzyme Can be used. A cross-linking reaction of about 10 minutes with 0.6% formaldehyde may be sufficient to inactivate RNase HII from Pyrococcus furiosus.

The crosslinked RNase H does not exhibit measurable endonuclease activity at about < RTI ID = 0.0 > 37 C. < / RTI > In some cases, a measurable partial reactivation of the crosslinked RNase H can occur at a temperature of about 50 캜, below the PCR heat denaturation temperature. To prevent inadvertent reactivation of such enzymes, it may be desirable to store and maintain the modified RNase H at temperatures below 50 ° C until reactivation.

Generally, the PCR should heat the amplification composition to about 95 ° C for each cycle to thermally denature the double-stranded target sequence, which also releases an inactivating factor from RNase H, , Or will fully recover.

RNase H can also be modified by acylating the lysine residue with an acylating agent, e. G. Dicarboxylic acid. Acylation of RNase H is accomplished by adding cis-aconitic anhydride to a solution of RNase H in acylation buffer, and incubating the resulting mixture at about 1 to 20 ° C for 5 to 30 hours ≪ / RTI > In one embodiment, the acylation may be carried out at about 3 to 8 占 폚 for 18 to 24 hours. The type of the acylation buffer solution is not particularly limited. In one embodiment, the acylation buffer has a pH of about 7.5 to about 9.0.

The activity of the acylated RNase H can be restored by lowering the pH of the amplification composition to less than about 7.0. For example, when Tris buffer is used as a buffer, the composition can be heated to about 95 DEG C to lower the pH from about 8.7 (25 DEG C) to about 6.5 (95 DEG C).

The duration of the heating step in the amplification reaction composition may vary depending on the modified RNase H, the buffer used in the PCR, and the like. In general, however, heating the amplification composition at 95 ° C. for about 30 seconds to 4 minutes may be sufficient to restore RNase H activity. In one embodiment, when using a commercially available buffer and one or more nonionic detergents, complete activity of Pyrococcus furiosus RNase HII is restored after 2 minutes of heating.

RNase H activity can be determined using methods well known in the art. For example, according to the first method, the unit activity is determined by an acid-solubilization of a certain number of moles of radiolabeled polyadenylic acid in the presence of a molar equivalent of polythymidylic acid, (See Epicenter Hybridase thermostable RNase HI). In a second method, the unit activity is defined as a specific increase in the relative fluorescence intensity of a reactant containing a molar equivalent of the probe and the complementary template DNA.

SNP Real-time detection

A labeled oligonucleotide probe can be used as a probe for real-time detection of the SNP in the target nucleic acid.

CataCleave ^TM oligonucleotide probes were first synthesized using DNA and RNA sequences complementary to sequences found in PCR amplicons containing a single nucleotide polymorphism (SNP). The probe may be synthesized, for example, using a FRET pair, for example fluorescein at one end of the probe and rhodamine quencher molecules at the other end. The probe may be synthesized to be substantially complementary to a target nucleic acid sequence comprising the position of the selected SNP.

In certain embodiments, the RNA sequence of the probe can be engineered to have a sequence complementary to the wild-type sequence.

In another embodiment, the RNA sequence of the probe can be engineered to have a sequence complementary to the SNP sequence.

In one embodiment, real-time nucleic acid amplification is performed in the presence of a thermostable nucleic acid polymerase, a RNase H activity, a pair of PCR amplification primers capable of hybridizing to a target polynucleotide comprising a SNP, and a labeled CataCleave ^TM oligonucleotide probe, Lt; / RTI > nucleotides. During the real-time PCR reaction, RNase H cleavage of the RNA: DNA heterodimer double-stranded probe formed between the CataCleave ^TM oligonucleotide probe and the SNP present in the PCR amplicon separates the fluorescent donor from the fluorescence quencher and the PCR amplicon, Resulting in real-time increase in probe fluorescence, corresponding to real-time detection of SNPs in DNA.

In certain embodiments, the RNA moiety of the probe comprises a wild-type sequence at a SNP position in the target DNA sequence. Thus, after hybridization of a probe with a PCR amplicon containing a SNP, the RNA: DNA heterozygous double stranded form with a single nucleotide mismatch at the SNP position can not be cleaved by RNase H.

In another embodiment, the RNA moiety of the probe comprises a complementary SNP sequence at the position of the SNP in the target DNA sequence. Thus, after hybridization of the PCR amplicon containing the SNP with the probe, an RNA: DNA heterozygous double strand is formed that does not have a mismatch at a SNP position that can be cleaved by RNase H activity.

Kit

The disclosure herein also provides a kit form comprising a package unit with one or more reagents for real-time detection of SNPs in the target nucleic acid. The kit may also include one or more of the following items: buffer, instructions for use and positive or negative control. The kit may comprise a container containing reagents mixed in suitable proportions to carry out the methods described herein. The reagent vessel preferably comprises the reagents in a unit quantity for omitting the measuring step when carrying out the subject method.

In addition, the kit may also comprise a thermostable polymerase, an RNase H, a primer selected to amplify a site comprising the SNP site, and a primer that anneals to the real-time PCR product to detect the SNP in accordance with the methods described herein But are not limited to, CataCleave ^TM oligonucleotide probes. The kit may comprise reagents for detecting SNPs in a single gene or loci of SNPs among two or more genes. In another embodiment, the kit reagents further comprise reagents for extracting genomic DNA or RNA from the biological sample. The kit reagent may also contain reagents for reverse transcriptase-PCR analysis, where applicable.

Any patents, patent applications, literature or other disclosures set forth herein are incorporated herein by reference in their entirety. Any content or portion thereof that conflicts with an existing definition, description, or other disclosure presented herein by reference is intended to be incorporated herein by reference to the extent that there is no conflict between the content being incorporated and the present disclosure Lt; / RTI >

Example

The following examples illustrate methods of using the RNase H enzyme compositions according to the present invention. The steps of the method described in these embodiments are not intended to be limiting. Additional objects and advantages of the invention other than those mentioned above will be apparent from the following examples, which are not intended to limit the scope of the invention.

Salmonella invA  Synthesis from genes SNP of Isothermal  detection

To test the ability of the CataCleave ^TM probe to distinguish single nucleotide sequence differences within the target DNA sequence, invA of Salmonella A synthetic single nucleotide polymorphism (SNP) was generated in the gene (SEQ ID NO: 33). The single nucleotide change resulted in the conversion of T to G at position 116 of Salmonella invA coding sequence (SEQ ID NO: 33). Two similar 19-nucleotide CataCleave ^™ probes, each double-labeled to generate a FRET pair, invA site to form a base pair. The wild-type specific probe inv-CCProbe2 (SEQ ID NO: 1) had perfect complementarity to the wild type of invA, and the SNP-specific probe inv-CCProbe2-2C (SEQ ID NO: 2) had complete complementarity with the mutation form of invA . The probes were designed such that the second of the four RNA bases of the CataCleave ^™ probe (at the 5 'end of the probe) forms a base pair at the SNP nucleotide position. Inv2-Target1 (SEQ ID NO: 3), which is complementary to two DNA oligonucleotides, a wild-type specific invA probe, and inv2-Target8 (SEQ ID NO: 4), which is complementary to SNP-specific invA probes, To assess the ability of the two probes to identify single nucleotide mismatches, an isothermal processing reaction was performed. The final concentration of each constituent in the reaction was 200 nM probe, 0.4 nM target oligonucleotide, 10 mM Tris acetate pH 8.6, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT and 2.5 u Hybridase thermal stability RNase GI (Hybridase thermostable RNase HI) (Epicenter). The reaction was incubated at 55 < 0 > C for 60 minutes, and fluorescence data were collected every minute. Figure 1 shows the fluorescence signal generated when inv-CCProbe2 (SEQ ID NO: 1) reacted with inv2-Target1 (SEQ ID NO: 3) or inv2-Target8 (SEQ ID NO: 4). When inv-CCProbe2 was incubated with inv2-Target1, the fluorescence signal increased linearly, indicating that RNase HI recognized and cleaved a probe that perfectly pairs with the oligonucleotide. When inv-CCProbe2 was incubated with inv2-Target8, fluorescence signals were scarcely generated, suggesting that the mismatched oligonucleotides were not nearly targeted to RNase HI.

Figure 2 shows the fluorescence signal generated when inv-CCProbe2-2C (SEQ ID NO: 2) was reacted with inv2-Target1 (SEQ ID NO: 3) or inv2-Target8 (SEQ ID NO: 4). Inv-CCProbe2-2C was cleaved by RNase HI when incubated with a perfectly matched inv2-Target8, indicated by an increase in fluorescence. When incubated with a wild-type invA target, fluorescence signals were hardly observed, indicating poor probe cleavage for mismatched pairs.

Salmonella invA Julian  Synthesis SNP Real time PCR  detection

Plasmid DNA containing the invA sequence of 267 nucleotides, including the wild type or the mutated form (described above), was synthesized. A 40 pg wild-type plasmid, a mutant plasmid, or a mixture of the two plasmids was used as a template for multiplex real-time PCR reactions including differently labeled probes complementary to wild-type or mutant sequences. The final concentration of each of the components was determined as follows: 800 nM forward primer Salmonella-F1 (SEQ ID NO: 5), 800 nM reverse primer sal-invR2 (SEQ ID NO: 6), 200 nM wild type specific probe inv-CCProbe2 nM SNP specific probe Inv-CCProbe2-2C (SEQ ID NO: 2), 80 uM each dNTP, 10 mM Tris acetate pH 8.6, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 2.5 u Platinum Taq DNA polymer Life Technologies and 2.5 u Hybridase Thermal Stability RNase HI (Epicenter). The PCR reaction was incubated at 95 ° C for 2 minutes to activate the hot start DNA polymerase, followed by 40 cycles of 95 ° C for 10 seconds, and 60 ° C for 20 seconds. Figure 3 shows the fluorescence signal generated during PCR from FAM labeled inv-CCProbe2 (SEQ ID NO: 1) and Figure 4 shows the fluorescence signal generated during PCR from TYE665 labeled inv-CCProbe2-2C (SEQ ID NO: 2) . The fluorescence curve for this PCR reaction indicates that each probe was able to detect the amplification of a perfectly complementary target and not amplification of the target containing one mismatch.

Small b Casein  Of the A1 and A2 alleles of the gene Isothermal  detection

In milk of cow there are two complete natural variants or forms of b-casein protein, called A2 and A1 b casein. The difference between A1 and A2b casein is a single amino acid at position 67. In the A1 variant, the base is T, and in the A2 variant the base is G. The A1 mutant b casein in milk is unique among all mammalian casein in that it has a histidine amino acid at this position. The wet of other species contains b casein, which can be thought of as an A2 analog, in that it has a proline amino acid at the same position in its b casein chain. Buffalo, yak, goat, and human wetting all contain A2 case-like casein.

In this example, two similar 19-nucleotide CataCleave ^™ probes, double-labeled to generate FRET pairs, were designed to form base pairs across the A1 / A2 SNP nucleotide positions of the bovine casein gene. A1-CCProbe2-RC (SEQ ID NO: 7) forms a complete base pair with the A1 allele and A2-CCProbe1-RC (SEQ ID NO: 8) forms a complete base pair with the A2 allele. The A1-CCProbe2-RC was designed such that the first of the four RNA bases of the CataCleave ^TM probe (at the 5 'end of the probe) forms a base pair at the SNP nucleotide position. Two DNA oligonucleotides were synthesized: A1-Target-RC (SEQ ID NO: 9) representing the A1 allele and A2-Target-RC (SEQ ID NO: 10) representing the A2 allele. To evaluate the ability of the two probes to identify single nucleotide differences, an isothermal reaction was performed using RNAse HI. Final concentrations of each of the constituents in the reaction were determined using a 200 nM probe, 0.4 nM target oligonucleotide, 10 mM Tris acetate pH 8.6, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT and 2.5 u Hybridase thermal stability RNase HI (Epicenter). Fluorescence data were collected every minute while the reaction was incubated at 55 ° C for 60 minutes. Figure 5 shows the fluorescence signal generated when Al-CCProbe2-RC (SEQ ID NO: 7) reacted with A1-Target-RC (SEQ ID NO: 9) or A2-Target-RC (SEQ ID NO: 10). When the A1-CCProbe2-RC was incubated with A1-Target-RC, the fluorescence signal increased linearly, indicating that RNase HI recognized and cleaved fully paired oligonucleotides. When A1-CCProbe2-RC was incubated with A2-Target-RC, fluorescent signals were scarcely generated, suggesting that mismatched oligonucleotides are poor targets for RNase HI. Figure 6 shows fluorescence signals generated when A2-CCProbe1-RC (SEQ ID NO: 8) was reacted with A1-Target-RC (SEQ ID NO: 9) or A2-Target-RC (SEQ ID NO: 10). The A2-CCProbe1-RC cleavage by RNase HI was performed when A2-CCProbe-RC was incubated with A2-Target-RC, but no cleavage occurred when incubated with A1-Target-RC.

Small b Casein  Real time of A1 and A2 alleles of genes PCR  detection

Three sets of dairy bull DNA were used for genotyping using CataCleave ^™ -based SNP detection. The three DNAs were previously analyzed for their genotypes by sequencing, and it was reported that they represent three possible genotypes of the casein gene, A1 / A1, A2 / A2 and A1 / A2. DNA was extracted from bull sperm as previously described by Heyen et al. (SEQ ID NO: 7) and A2-CCProbe1-RC (SEQ ID NO: 8), which are differentially expressed, from the genomic DNA of the A1 / A2 genotype, A2 / A2 genotype or A1 / , As a template for multiplex real-time PCR reactions. The final concentration of each component was determined using the following primers: 800 nM forward primer A2D-F (SEQ ID NO: 11), 800 nM reverse primer A2D-R-150 (SEQ ID NO: 12), 200 nM A1-CCProbe2- 10 mM Tris acetate pH 8.6, 50 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 2.5 u Platinum Taq DNA polymerase (manufactured by Life Technologies Inc.), 200 nM A2-CCProbe-RC ) And 2.5 u hybridization thermal stability RNase HI (Epicenter). The PCR reaction was incubated at 95 C for 2 minutes to activate the hot start DNA polymerase followed by 40 cycles of 95 ° C for 10 seconds and 60 ° C for 30 seconds. Figure 7 shows the fluorescence signal generated during PCR from TYE563-labeled A1-CCProbe2-RC (SEQ ID NO: 7) and Figure 8 shows the signal generated during PCR from TYE665 labeled A2-CCProbe1-RC (SEQ ID NO: 8). The fluorescence curve for the PCR reaction suggests that each probe is capable of detecting amplification of a perfectly complementary target and not amplification of a target containing a single mismatch.

Any patents, patent applications, literature or other disclosures set forth herein are incorporated herein by reference in their entirety. Any content, or any portion thereof, that is inconsistent with an existing definition, description, or other disclosure incorporated by reference herein, but which is incorporated herein by reference, is incorporated herein to the extent that no conflicts of circumstances arise between the integrated content and the present disclosure .

See the attached sequence listing (SEQ ID NOS: 1-33).

<110> Samsung Techwin Co. Ltd. <120> REAL TIME PCR DETECTION OF SINGLE NUCLEOTIDE POLYMORPHISMS <130> PN089052 &Lt; 150 > US61 / 390,701 <151> 2010-10-07 <150> US13 / 158,593 <151> 2011-06-13 <160> 33 <170> PatentIn version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (1) <223> 5 '6-carboxyfluorescein donor chromophore <220> <221> misc_feature <222> (1) 56-FAM: 5 '6-carboxyfluorescein donor chromophore <220> <221> misc_feature <222> (1) (7) <223> DNA sequence <220> <221> misc_feature &Lt; 222 > (8) <223> RNA sequence <220> <221> misc_feature <222> (12) (19) <223> DNA sequence <220> <221> misc_feature <222> (19) <223> 3IABlkFQ: 3 'Iowa Black FQ Quencher <400> 1 cgatcaggaa atcaaccag 19 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (1) <223> 5 'TYE563 fluorescent dye <220> <221> misc_feature <222> (1) (7) <223> DNA sequence <220> <221> misc_feature &Lt; 222 > (8) <223> RNA sequence <220> <221> misc_feature <222> (12) (19) <223> DNA sequence <220> <221> misc_feature <222> (19) <223> 3 'IABlkFQ: 3 "Iowa Black FQ quencher <400> 2 cgatcaggca atcaaccag 19 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 3 cacactggtt gatttcctga tcgcaca 27 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 4 cacactggtt gattgcctga tcgcaca 27 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 5 tcgtcattcc attacctacc 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 6 tactgatcga taatgccaga cgaa 24 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (1) <223> 5TYE563; 5: TYE 563 fluorescent dye <220> <221> misc_feature <222> (1) (8) <223> DNA sequence <220> <221> misc_feature <222> (9) <223> RNA sequence <220> <221> misc_feature <222> (13). (18) <223> DNA sequence <220> <221> misc_feature <222> (18) <223> 3IAbRQSp: 3 'Iowa Black RQ-Sp Dark Quecher <220> <221> misc_RNA <222> (11) <223> n is uracil <400> 7 ggcccatcca naacagcc 18 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <220> <221> misc_feature <222> (1) &Lt; 223 > 5TYE665: 5 'TYE665 fluorescent dye <220> <221> misc_feature <222> (1) (9) <223> DNA sequence <220> <221> misc_feature &Lt; 222 > (10) <223> RNA sequence <220> <221> misc_feature <222> (14). (18) <223> DNA sequence <220> <221> misc_feature <222> (18) <223> 3IAbRQSp: 3 'Iowa Black RQ-Sp Dark Quencher <220> <221> misc_RNA <222> (11) <223> n is uracil <400> 8 ggcccatccc naacagcc 18 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 9 gagaggctgt tatggatggg ccgaga 26 <210> 10 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 10 gagaggctgt tagggatggg ccgaga 26 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 11 gatgaactcc aggataaaat ccacc 25 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic oligonucleotide <400> 12 tacttcaggc tgaaggaaag g 21 <210> 13 <211> 224 <212> PRT <213> Pyrococcus furiosus <400> 13 Met Lys Ile Gly Gly Ile Asp Glu Ala Gly Arg Gly Pro Ala Ile Gly   1 5 10 15 Pro Leu Val Val Ala Thr Val Val Val Asp Glu Lys Asn Ile Glu Lys              20 25 30 Leu Arg Asn Ile Gly Val Lys Asp Ser Lys Gln Leu Thr Pro His Glu          35 40 45 Arg Lys Asn Leu Phe Ser Gln Ile Thr Ser Ile Asp Asp Tyr Lys      50 55 60 Ile Val Ile Val Ser Pro Glu Glu Ile Asp Asn Arg Ser Gly Thr Met  65 70 75 80 Asn Glu Leu Glu Val Glu Lys Phe Ala Leu Ala Leu Asn Ser Leu Gln                  85 90 95 Ile Lys Pro Ala Leu Ile Tyr Ala Asp Ala Ala Asp Val Asp Ala Asn             100 105 110 Arg Phe Ala Ser Leu Ile Glu Arg Arg Leu Asn Tyr Lys Ala Lys Ile         115 120 125 Ile Ala Glu His Lys Ala Asp Ala Lys Tyr Pro Val Val Ser Ala Ala     130 135 140 Ser Ile Leu Ala Lys Val Val Arg Asp Glu Glu Ile Glu Lys Leu Lys 145 150 155 160 Lys Gln Tyr Gly Asp Phe Gly Ser Gly Tyr Pro Ser Asp Pro Lys Thr                 165 170 175 Lys Lys Trp Leu Glu Glu Tyr Tyr Lys Lys His Asn Ser Phe Pro Pro             180 185 190 Ile Val Arg Arg Thr Trp Glu Thr Val Arg Lys Ile Glu Glu Ser Ile         195 200 205 Lys Ala Lys Lys Ser Gln Leu Thr Leu Asp Lys Phe Phe Lys Lys Pro     210 215 220 <210> 14 <211> 220 <212> PRT <213> Pyrococcus horikoshi <400> 14 Met Lys Val Ala Gly Val Asp Glu Ala Gly Arg Gly Pro Val Ile Gly   1 5 10 15 Pro Leu Val Ile Gly Val Ala Val Ile Asp Glu Lys Asn Ile Glu Arg              20 25 30 Leu Arg Asp Ile Gly Val Lys Asp Ser Lys Gln Leu Thr Pro Gly Gln          35 40 45 Arg Glu Lys Leu Phe Ser Lys Leu Ile Asp Ile Leu Asp Asp Tyr Tyr      50 55 60 Val Leu Leu Val Thr Pro Lys Glu Ile Asp Glu Arg His His Ser Met  65 70 75 80 Asn Glu Leu Glu Ala Glu Lys Phe Val Val Ala Leu Asn Ser Leu Arg                  85 90 95 Ile Lys Pro Gln Lys Ile Tyr Val Asp Ser Ala Asp Val Asp Pro Lys             100 105 110 Arg Phe Ala Ser Leu Ile Lys Ala Gly Leu Lys Tyr Glu Ala Thr Val         115 120 125 Ile Ala Glu His Lys Ala Asp Ala Lys Tyr Glu Ile Val Ser Ala Ala     130 135 140 Ser Ile Ile Ala Lys Val Thr Arg Asp Arg Glu Ile Glu Lys Leu Lys 145 150 155 160 Gln Lys Tyr Gly Glu Phe Gly Ser Gly Tyr Pro Ser Asp Pro Arg Thr                 165 170 175 Lys Glu Trp Leu Glu Glu Tyr Tyr Lys Gln Tyr Gly Asp Phe Pro Pro             180 185 190 Ile Val Arg Arg Thr Trp Glu Thr Ala Arg Lys Ile Glu Glu Arg Phe         195 200 205 Arg Lys Asn Gln Leu Thr Leu Asp Lys Phe Leu Lys     210 215 220 <210> 15 <211> 228 <212> PRT <213> Thermococcus kodakarensis <400> 15 Met Lys Ile Ala Gly Ile Asp Glu Ala Gly Arg Gly Pro Val Ile Gly   1 5 10 15 Pro Met Val Ile Ala Val Val Val Asp Glu Asn Ser Leu Pro Lys              20 25 30 Leu Glu Glu Leu Lys Val Arg Asp Ser Lys Lys Leu Thr Pro Lys Arg          35 40 45 Arg Glu Lys Leu Phe Asn Glu Ile Leu Gly Val Leu Asp Asp Tyr Val      50 55 60 Ile Leu Glu Leu Pro Pro Asp Val Ile Gly Ser Arg Glu Gly Thr Leu  65 70 75 80 Asn Glu Phe Glu Val Glu Asn Phe Ala Lys Ala Leu Asn Ser Leu Lys                  85 90 95 Val Lys Pro Asp Val Ile Tyr Ala Asp Ala Ala Asp Val Asp Glu Glu             100 105 110 Arg Phe Ala Arg Glu Leu Gly Glu Arg Leu Asn Phe Glu Ala Glu Val         115 120 125 Val Ala Lys His Lys Ala Asp Asp Ile Phe Pro Val Val Ser Ala Ala     130 135 140 Ser Ile Leu Ala Lys Val Thr Arg Asp Arg Ala Val Glu Lys Leu Lys 145 150 155 160 Glu Glu Tyr Gly Glu Ile Gly Ser Gly Tyr Pro Ser Asp Pro Arg Thr                 165 170 175 Arg Ala Phe Leu Glu Asn Tyr Tyr Arg Glu His Gly Glu Phe Pro Pro             180 185 190 Ile Val Arg Lys Gly Trp Lys Thr Leu Lys Lys Ile Ala Glu Lys Val         195 200 205 Glu Ser Glu Lys Lys Ala Glu Glu Arg Gln Ala Thr Leu Asp Arg Tyr     210 215 220 Phe Arg Lys Val 225 <210> 16 <211> 211 <212> PRT <213> Archaeoglobus profundus <400> 16 Met Ile Ala Gly Ile Asp Glu Ala Gly Lys Gly Pro Val Ile Gly Pro   1 5 10 15 Leu Val Ile Cys Gly Val Leu Cys Asp Glu Glu Thr Val Glu Tyr Leu              20 25 30 Lys Ser Val Gly Val Lys Asp Ser Lys Lys Leu Asp Arg Arg Lys Arg          35 40 45 Glu Glu Leu Tyr Asn Ile Ile Lys Ser Leu Cys Lys Val Lys Val Leu      50 55 60 Lys Ile Ser Val Glu Asp Leu Asn Arg Leu Met Glu Tyr Met Ser Ile  65 70 75 80 Asn Glu Ile Leu Lys Arg Ala Tyr Val Glu Ile Ile Arg Ser Leu Met                  85 90 95 Pro Lys Val Val Tyr Ile Asp Cys Pro Asp Ile Asn Val Glu Arg Phe             100 105 110 Lys His Glu Ile Glu Glu Arg Thr Gly Val Glu Val Phe Ala Ser His         115 120 125 Lys Ala Asp Glu Ile Tyr Pro Ile Val Ser Ile Ala Ser Ile Val Ala     130 135 140 Lys Val Glu Arg Asp Phe Glu Ile Asp Lys Leu Lys Lys Ile Tyr Gly 145 150 155 160 Asp Phe Gly Ser Gly Tyr Pro Ser Asp Leu Arg Thr Ile Glu Phe Leu                 165 170 175 Arg Ser Tyr Leu Arg Glu His Lys Ser Phe Pro Pro Ile Val Arg Lys             180 185 190 Arg Trp Lys Thr Leu Lys Arg Leu Thr Thr His Thr Leu Ser Asp Phe         195 200 205 Phe Glu Val     210 <210> 17 <211> 205 <212> PRT <213> Archaeoglobus fulgidis <400> 17 Met Lys Ala Gly Ile Asp Glu Ala Gly Lys Gly Cys Val Ile Gly Pro   1 5 10 15 Leu Val Val Ala Gly Val Ala Cys Ser Asp Glu Asp Arg Leu Arg Lys              20 25 30 Leu Gly Val Lys Asp Ser Lys Lys Leu Ser Gln Gly Arg Arg Glu Glu          35 40 45 Leu Ala Glu Glu Ile Arg Lys Ile Cys Arg Thr Glu Val Leu Lys Val      50 55 60 Ser Pro Glu Asn Leu Asp Glu Arg Met Ala Ala Lys Thr Ile Asn Glu  65 70 75 80 Ile Leu Lys Glu Cys Tyr Ala Glu Ile Ile Leu Arg Leu Lys Pro Glu                  85 90 95 Ile Ala Tyr Val Asp Ser Pro Asp Val Ile Pro Glu Arg Leu Ser Arg             100 105 110 Glu Leu Glu Glu Ile Thr Gly Leu Arg Val Val Ala Glu His Lys Ala         115 120 125 Asp Glu Lys Tyr Pro Leu Val Ala Ala Ser Ile Ile Ala Lys Val     130 135 140 Glu Arg Glu Arg Glu Ile Glu Arg Leu Lys Glu Lys Phe Gly Asp Phe 145 150 155 160 Gly Ser Gly Tyr Ala Ser Asp Pro Arg Thr Arg Glu Val Leu Lys Glu                 165 170 175 Trp Ile Ala Ser Gly Arg Ile Pro Ser Cys Val Arg Met Arg Trp Lys             180 185 190 Thr Val Ser Asn Leu Arg Gln Lys Thr Leu Asp Asp Phe         195 200 205 <210> 18 <211> 233 <212> PRT <213> Thermococcus celer <400> 18 Leu Lys Leu Ala Gly Ile Asp Glu Ala Gly Arg Gly Pro Val Ile Gly   1 5 10 15 Pro Met Val Ile Ala Val Val Leu Asp Glu Lys Asn Val Pro Lys              20 25 30 Leu Arg Asp Leu Gly Val Arg Asp Ser Lys Lys Leu Thr Pro Lys Arg          35 40 45 Arg Glu Arg Leu Phe Asn Asp Ile Ile Lys Leu Leu Asp Asp Tyr Val      50 55 60 Ile Leu Glu Leu Trp Pro Glu Glu Ile Asp Ser Arg Gly Gly Thr Leu  65 70 75 80 Asn Glu Leu Glu Val Glu Arg Phe Val Glu Ala Leu Asn Ser Leu Lys                  85 90 95 Val Lys Pro Asp Val Val Tyr Ile Asp Ala Ala Asp Val Lys Glu Gly             100 105 110 Arg Phe Gly Glu Glu Ile Lys Glu Arg Leu Asn Phe Glu Ala Lys Ile         115 120 125 Val Ser Glu His Arg Ala Asp Asp Lys Phe Leu Pro Val Ser Ser Ala     130 135 140 Ser Ile Leu Ala Lys Val Thr Arg Asp Arg Ala Ile Glu Lys Leu Lys 145 150 155 160 Glu Lys Tyr Gly Glu Ile Gly Ser Gly Tyr Pro Ser Asp Pro Arg Thr                 165 170 175 Arg Glu Phe Leu Glu Asn Tyr Tyr Arg Gln His Gly Glu Phe Pro Pro             180 185 190 Val Val Arg Arg Ser Trp Lys Thr Leu Arg Lys Ile Glu Glu Lys Leu         195 200 205 Arg Lys Glu Ala Gly Ser Lys Asn Pro Glu Asn Ser Lys Glu Lys Gly     210 215 220 Gln Thr Ser Leu Asp Val Phe Leu Arg 225 230 <210> 19 <211> 224 <212> PRT <213> Thermococcus litoralis <400> 19 Met Lys Leu Gly Gly Ile Asp Glu Ala Gly Arg Gly Pro Val Ile Gly   1 5 10 15 Pro Leu Val Ile Ala Ala Val Val Val Asp Glu Ser Arg Met Gln Glu              20 25 30 Leu Glu Ala Leu Gly Val Lys Asp Ser Lys Lys Leu Thr Pro Lys Arg          35 40 45 Arg Glu Glu Leu Phe Glu Glu Ile Val Gln Ile Val Asp Asp His Val      50 55 60 Ile Ile Gln Leu Ser Pro Glu Glu Ile Asp Gly Arg Asp Gly Thr Met  65 70 75 80 Asn Glu Leu Glu Ile Glu Asn Phe Ala Lys Ala Leu Asn Ser Leu Lys                  85 90 95 Val Lys Pro Asp Val Leu Tyr Ile Asp Ala Ala Asp Val Lys Glu Lys             100 105 110 Arg Phe Gly Asp Ile Ile Gly Glu Arg Leu Ser Phe Ser Pro Lys Ile         115 120 125 Ile Ala Glu His Lys Ala Asp Ser Lys Tyr Ile Pro Val Ala Ala Ala     130 135 140 Ser Ile Leu Ala Lys Val Thr Arg Asp Arg Ala Ile Glu Lys Leu Lys 145 150 155 160 Glu Leu Tyr Gly Glu Ile Gly Ser Gly Tyr Pro Ser Asp Pro Asn Thr                 165 170 175 Arg Arg Phe Leu Glu Glu Tyr Tyr Lys Ala His Gly Glu Phe Pro Pro             180 185 190 Ile Val Arg Lys Ser Trp Lys Thr Leu Arg Lys Ile Glu Glu Lys Leu         195 200 205 Lys Ala Lys Lys Thr Gln Pro Thr Ile Leu Asp Phe Leu Lys Lys Pro     210 215 220 <210> 20 <211> 16 <212> PRT <213> Pyrococcus furiosis <400> 20 Gly Ile Asp Glu Ala Gly Arg Gly Pro Ala Ile Gly Pro Leu Val Val   1 5 10 15 <210> 21 <211> 12 <212> PRT <213> Pyrococcus furiosis <400> 21 Leu Arg Asn Ile Gly Val Lys Asp Ser Lys Gln Leu   1 5 10 <210> 22 <211> 19 <212> PRT <213> Pyrococcus furiosis <400> 22 His Lys Ala Asp Ala Lys Tyr Pro Val Val Ser Ala Ala Ser Ile Leu   1 5 10 15 Ala Lys Val             <210> 23 <211> 16 <212> PRT <213> Pyrococcus furiosis <400> 23 Lys Leu Lys Lys Gln Tyr Gly Asp Phe Gly Ser Gly Tyr Pro Ser Asp   1 5 10 15 <210> 24 <211> 174 <212> PRT <213> Haemophilus influenzae <400> 24 Met Phe Asn Leu Ser Leu Ser Ile Lys Ile Pro Ala Ile Leu His Asn   1 5 10 15 Asn Leu Phe Val Met Gln Lys Gln Ile Glu Ile Phe Thr Asp Gly Ser              20 25 30 Cys Leu Gly Asn Pro Gly Ala Gly Gly Gly Ile Gly Ala Val Leu Arg Tyr          35 40 45 Lys Gln His Glu Lys Met Leu Ser Lys Gly Tyr Phe Lys Thr Thr Asn      50 55 60 Asn Arg Met Glu Leu Arg Ala Val Ile Glu Ala Leu Asn Thr Leu Lys  65 70 75 80 Glu Pro Cys Leu Ile Thr Leu Tyr Ser Asp Ser Gln Tyr Met Lys Asn                  85 90 95 Gly Ile Thr Lys Trp Ile Phe Asn Trp Lys Lys Asn Asn Trp Lys Ala             100 105 110 Ser Ser Gly Lys Pro Val Lys Asn Gln Asp Leu Trp Ile Ala Leu Asp         115 120 125 Glu Ser Ile Gln Arg His Lys Ile Asn Trp Gln Trp Val Lys Gly His     130 135 140 Ala Gly His Arg Glu Asn Glu Ile Cys Asp Glu Leu Ala Lys Lys Gly 145 150 155 160 Ala Glu Asn Pro Thr Leu Glu Asp Met Gly Tyr Phe Glu Glu                 165 170 <210> 25 <211> 166 <212> PRT <213> Thermus thermophilus <400> 25 Met Asn Pro Ser Pro Arg Lys Arg Val Ala Leu Phe Thr Asp Gly Ala   1 5 10 15 Cys Leu Gly Asn Pro Gly Pro Gly Gly Trp Ala Ala Leu Leu Arg Phe              20 25 30 His Ala His Glu Lys Leu Leu Ser Gly Gly Glu Ala Cys Thr Thr Asn          35 40 45 Asn Arg Met Glu Leu Lys Ala Ala Ile Glu Gly Leu Lys Ala Leu Lys      50 55 60 Glu Pro Cys Glu Val Asp Leu Tyr Thr Asp Ser His Tyr Leu Lys Lys  65 70 75 80 Ala Phe Thr Glu Gly Trp Leu Glu Gly Trp Arg Lys Arg Gly Trp Arg                  85 90 95 Thr Ala Glu Gly Lys Pro Val Lys Asn Arg Asp Leu Trp Glu Ala Leu             100 105 110 Leu Leu Ala Met Ala Pro His Arg Val Phe His His Val Lys Gly         115 120 125 His Thr Gly His Pro Glu Asn Glu Arg Val Asp Arg Glu Ala Arg Arg     130 135 140 Gln Ala Gln Ser Gln Ala Lys Thr Pro Cys Pro Pro Arg Ala Pro Thr 145 150 155 160 Leu Phe His Glu Glu Ala                 165 <210> 26 <211> 161 <212> PRT <213> Thermus aquaticus <400> 26 Met Ser Leu Pro Leu Lys Arg Val Asp Leu Phe Thr Asp Gly Ala Cys   1 5 10 15 Leu Gly Asn Pro Gly Pro Gly Gly Trp Ala Leu Leu Arg Tyr Gly              20 25 30 Ser Gln Glu Lys Leu Leu Ser Gly Gly Glu Pro Cys Thr Thr Asn Asn          35 40 45 Arg Met Glu Leu Arg Ala Leu Glu Gly Leu Leu Ala Leu Arg Glu      50 55 60 Pro Cys Gln Val His Leu His Thr Asp Ser Gln Tyr Leu Lys Arg Ala  65 70 75 80 Phe Ala Glu Gly Trp Val Glu Arg Trp Gln Arg Asn Gly Trp Arg Thr                  85 90 95 Ala Glu Gly Lys Pro Val Lys Asn Gln Asp Leu Trp Gln Ala Leu Leu             100 105 110 Lys Ala Met Glu Gly His Glu Val Ala Phe His Phe Val Glu Gly His         115 120 125 Ser Gly His Pro Glu Asn Glu Arg Val Asp Arg Glu Ala Arg Arg Gln     130 135 140 Ala Lys Ala Gln Pro Gln Val Pro Cys Pro Pro Lys Glu Ala Thr Leu 145 150 155 160 Phe     <210> 27 <211> 146 <212> PRT <213> Salmonella enterica <400> 27 Met Leu Lys Gln Val Glu Ile Phe Thr Asp Gly Ser Cys Leu Gly Asn   1 5 10 15 Pro Gly Pro Gly Gly Tyr Gly Ala Ile Leu Arg Tyr Arg Gly His Glu              20 25 30 Lys Thr Phe Ser Glu Gly Tyr Thr Leu Thr Thr Asn Asn Arg Met Glu          35 40 45 Leu Met Ala Ile Val Ala Leu Glu Ala Leu Lys Glu His Cys Glu      50 55 60 Val Thr Leu Ser Thr Asp Ser Gln Tyr Val Arg Gln Gly Ile Thr Gln  65 70 75 80 Trp Ile His Asn Trp Lys Lys Arg Gly Trp Lys Thr Ala Glu Lys Lys                  85 90 95 Pro Val Lys Asn Val Asp Leu Trp Lys Arg Leu Asp Ala Ala Leu Gly             100 105 110 Gln His Gln Ile Lys Trp Val Trp Val Lys Gly His Ala Gly His Pro         115 120 125 Glu Asn Glu Arg Cys Asp Glu Leu Ala Arg Ala Ala Met Asn Pro     130 135 140 Thr Gln 145 <210> 28 <211> 146 <212> PRT <213> Agrobacterium tumefaciens <400> 28 Met Lys His Val Asp Ile Phe Thr Asp Gly Ala Cys Ser Gly Asn Pro   1 5 10 15 Gly Pro Gly Gly Trp Gly Ala Val Leu Arg Tyr Gly Glu Thr Glu Lys              20 25 30 Glu Leu Ser Gly Gly Glu Ala Asp Thr Thr Asn Asn Arg Met Glu Leu          35 40 45 Leu Ala Ile Ser Ala Leu Asn Ala Leu Lys Ser Pro Cys Glu Val      50 55 60 Asp Leu Tyr Thr Asp Ser Ala Tyr Val Lys Asp Gly Ile Thr Lys Trp  65 70 75 80 Ile Phe Gly Trp Lys Lys Lys Lys Gly Trp Lys Thr Ala Asp Asn Lys Pro                  85 90 95 Val Lys Asn Val Glu Leu Trp Gln Ala Leu Glu Ala Ala Gln Glu Arg             100 105 110 His Lys Val Thr Leu His Trp Val Lys Gly His Ala Gly His Pro Glu         115 120 125 Asn Glu Arg Ala Asp Glu Leu Ala Arg Lys Gly Met Glu Pro Phe Lys     130 135 140 Arg Arg 145 <210> 29 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 29 Lys Xaa Val Xaa Leu Phe Thr Asp Gly Xaa Cys Xaa Gly Xaa Pro Gly   1 5 10 15 Xaa Gly Gly Xaa Ala Leu Leu Arg Tyr              20 25 <210> 30 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 30 Thr Asn Asn Arg Met Glu Leu   1 5 <210> 31 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 31 Lys Pro Val Lys Asn   1 5 <210> 32 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 32 Phe Val Lys Gly His Xaa Gly His Xaa Glu Asn Glu   1 5 10 <210> 33 <211> 1950 <212> DNA <213> Salmonella enterica <400> 33 aacagtgctc gtttacgacc tgaattactg attctggtac taatggtgat gatcatttct 60 atgttcgtca ttccattacc tacctatctg gttgatttcc tgatcgcact gaatatcgta 120 ctggcgatat tggtgtttat ggggtcgttc tacattgaca gaatcctcag tttttcaacg 180 tttcctgcgg tactgttaat taccacgctc tttcgtctgg cattatcgat cagtaccagc 240 cgtcttatct tgattgaagc cgatgccggt gaaattatcg ccacgttcgg gcaattcgtt 300 attggcgata gcctggcggt gggttttgtt gtcttctcta ttgtcaccgt ggtccagttt 360 atcgttatta ccaaaggttc agaacgcgtc gcggaagtcg cggcccgatt ttctctggat 420 ggtatgcccg gtaaacagat gagtattgat gccgatttga aggccggtat tattgatgcg 480 gatgctgcgc gcgaacggcg aagcgtactg gaaagggaaa gccagcttta cggttccttt 540 gacggtgcga tgaagtttat caaaggtgac gctattgccg gcatcattat tatctttgtg 600 aactttattg gcggtatttc ggtggggatg acccgccatg gtatggattt gtcctccgct 660 ctgtctactt ataccatgct gaccattggt gatggtcttg tcgcccagat ccccgcattg 720 ttgattgcga ttagtgccgg ttttatcgtg actcgcgtaa atggcgatag cgataatatg 780 gggcggaata tcatgacgca gctgttgaac aacccatttg tattggttgt tacggctatt 840 ttgaccattt caatgggaac tctgccggga ttcccgctgc cggtatttgt tattttatcg 900 gtggttttaa gcgtactctt ctattttaaa ttccgtgaag caaaacgtag cgccgccaaa 960 cctaaaacca gcaaaggcga gcagccgctt agtattgagg aaaaagaagg gtcgtcgttg 1020 ggactgattg gcgatctcga taaagtctct acagagaccg taccgttgat attacttgtg 1080 ccgaagagcc ggcgtgaaga tctggaaaaa gctcaacttg cggagcgtct acgtagtcag 1140 ttctttattg attatggcgt gcgcctgccg gaagtattgt tacgcgatgg cgagggcctg 1200 gacgataaca gcatcgtatt gttgattaat gagatccgtg ttgaacaatt tacggtctat 1260 tttgatttga tgcgagtggt aaattattcc gatgaagtcg tgtcctttgg tattaatcca 1320 acaatccatc agcaaggtag cagtcagtat ttctgggtaa cgcatgaaga gggggagaaa 1380 ctccgggagc ttggctatgt gttgcggaac gcgcttgatg agctttacca ctgtctggcg 1440 gtgaccgtgg cgcgcaacgt caatgaatat ttcggtattc aggaaacaaa acatatgctg 1500 gaccaactgg aagcgaaatt tcctgattta cttaaagaag tgctcagaca tgccacggta 1560 caacgtatat ctgaagtttt gcagcgttta ttaagcgaac gtgtttccgt gcgtaatatg 1620 aaattaatta tggaagcgct cgcattgtgg gcgccaagag aaaaagatgt cattaacctt 1680 gtagagcata ttcgtggagc aatggcgcgt tatatttgtc ataaattcgc caatggcggc 1740 gaattacgag cagtaatggt atctgctgaa gttgaggatg ttattcgcaa agggatccgt 1800 cagacctctg gcagtacctt cctcagcctt gacccggaag cctccgctaa tttgatggat 1860 ctcattacac ttaagttgga tgatttattg attgcacata aagatcttgt cctccttacg 1920 tctgtcgatg tccgtcgatt tattaagaaa 1950

Claims (27)

a) providing a sample to be tested for the presence of a target DNA having polymorphism;
b) providing an amplification primer pair capable of annealing to the target DNA, wherein the first amplification primer anneals upstream of a location of polymorphism, and the second amplification Wherein the primer anneals to a downstream of the polymorphic site;
c) providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is entirely complementary to a selected region of the target DNA sequence comprising the polymorphism, ) The DNA nucleic acid sequence of the probe is substantially complimentary to the DNA sequence adjacent to the selected region of the target DNA sequence;
and d) amplifying polymerase activity, amplification buffering conditions, and conditions, such that the RNA sequence in the probe is capable of forming an RNA: DNA heteroduplex with a complementary DNA sequence in the PCR fragment comprising the polymorphism an amplification buffer; Amplifying a PCR fragment between said first and second amplification primers in the presence of RNase H activity and a probe; And
e) detecting a real-time increase in signal emission from the label on the probe,
Wherein the increase in the signal indicates the presence of a polymorphism in the target DNA. a) providing a sample to be tested for the presence of a target DNA having a polymorphism;
b) providing an amplification primer pair capable of annealing to the target DNA, wherein the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position;
c) providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected portion of the target DNA sequence comprising the wild-type DNA sequence at the polymorphic position, Wherein the DNA nucleic acid sequence of the probe is substantially complementary to the DNA sequence adjacent to the selected region of the target DNA sequence;
d) an amplified polymerase activity, an amplification buffer, and an amplification buffer, under conditions such that the RNA sequence in the probe is capable of forming an RNA: DNA heterodimer double strand with a complementary DNA sequence in the PCR fragment comprising the polymorphism; Amplifying the PCR fragment between the first and second amplification primers in the presence of RNase H activity and a probe; And
e) detecting a real-time decrease in signal emission from the label on the probe,
Wherein the reduction of the signal is indicative of the presence of a polymorphism in the target DNA. a) providing a sample to be tested for the presence of a target RNA having a polymorphism;
b) providing an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position ;
c) providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected region of the cDNA sequence comprising the polymorphism, is substantially complementary to a DNA sequence adjacent to a selected region of the cDNA sequence;
d) reverse transcriptase activity, amplified polymerase activity, reverse transcriptase-PCR buffer; RNase H activity and a probe and wherein the RNA sequence in said probe is capable of forming an RNA: DNA heterodimer double strand with a complementary sequence in an RT-PCR DNA fragment comprising said polymorphism, Amplifying a reverse transcriptase-PCR fragment between the second amplification primers; And
e) detecting a real-time increase in signal emission from the label on the probe,
Wherein the increase in the signal indicates the presence of a polymorphism in the RNA target. 4. The method of claim 1 or 3, wherein a real-time increase in signal emission from the label on the probe results from RNase H cleavage of the RNA sequence of the RNA: DNA heterodimer double-stranded probe. 3. The method of claim 1, wherein the RNA nucleic acid sequence of the probe is complementary to the polymorphism in the target DNA. 4. The method of claim 1 or 3, wherein the polymorphism is a single nucleotide polymorphism (SNP). 4. The method of claim 1 or 3, wherein the DNA and RNA sequences of the probes are covalently linked. 4. The method of claim 1 or 3, wherein the detectable label on the probe is a fluorescent label. 9. The method of claim 8, wherein the fluorescent label comprises a FRET pair. 4. The method of claim 1 or 3, wherein the PCR fragment is attached to a solid support. 4. The method of claim 3, wherein the RNA nucleic acid sequence of the probe comprises an RNA sequence complementary to the cDNA of the polymorphic position in the target RNA. 4. The method of claim 3, wherein the RNA target is an mRNA transcript. 4. The method of claim 3, wherein the RNase H activity is an activity of hot start, thermostable RNase H. a) providing a sample to be tested for the presence of an RNA target having a polymorphism;
b) providing an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position ;
c) providing a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected portion of the cDNA comprising the wild-type DNA sequence at the polymorphic position, Wherein the DNA nucleic acid sequence is substantially complementary to the DNA sequence adjacent to the selected site of the cDNA;
d) reverse transcriptase activity, amplified polymerase activity, reverse transcriptase-PCR buffer; RNase H activity and a probe and wherein the RNA sequence in said probe is capable of forming an RNA: DNA heterodimer double strand with a complementary sequence in an RT-PCR DNA fragment comprising said polymorphism, Amplifying a reverse transcriptase-PCR fragment between the second amplification primers; And
e) detecting a real-time decrease in signal emission from the label on the probe,
Wherein the reduction of the signal is indicative of the presence of a polymorphism in the RNA target. a) an amplification primer pair capable of annealing to target DNA, wherein the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position;
b) a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected region of the target DNA sequence comprising the polymorphism, and wherein the DNA nucleic acid sequence of the probe is the target DNA sequence The probe being substantially complementary to a DNA sequence adjacent to a selected site of the probe;
c) amplified polymerase activity, amplification buffer; RTI ID = 0.0 &gt; RNase &lt; / RTI &gt; H activity. a) an amplification primer pair capable of annealing to target DNA, wherein the first amplification primer anneals upstream of the polymorphic position and the second amplification primer anneals downstream of the polymorphic position;
b) a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to a selected region of the target DNA sequence comprising the wild-type DNA sequence at the polymorphic position, Is substantially complementary to a DNA sequence adjacent to a selected region of the target DNA sequence;
c) amplified polymerase activity, amplification buffer; RTI ID = 0.0 &gt; RNase &lt; / RTI &gt; H activity. a) an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer anneals upstream of the polymorphic sequence position and the second amplification primer anneals downstream of the polymorphic sequence position;
b) a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to the selected site of the cDNA comprising the polymorphism, and the DNA nucleic acid sequence of the probe is selected A probe that is substantially complementary to an adjacent DNA sequence; And
c) reverse transcriptase activity, amplified polymerase activity, reverse transcriptase-PCR buffer; And a RNase H activity. 16. The kit of claim 15, wherein the RNA nucleic acid sequence of the probe comprises a sequence complementary to the polymorphism in the target DNA. 18. The kit of claim 15 or 17, wherein the polymorphism is a single nucleotide polymorphism (SNP). 18. The kit of claim 15 or 17 wherein the DNA and RNA sequences of the probe are covalently linked. 18. The kit of claim 15 or 17, wherein the detectable label on the probe is a fluorescent label. 22. The kit of claim 21, wherein the fluorescent label is a FRET pair. 16. The kit of claim 15, wherein the probe or PCR fragment is attached to a solid support. 18. The kit of claim 17, wherein the RNA nucleic acid sequence of the probe comprises a sequence complementary to a cDNA of a polymorphic position in the target RNA. 18. The kit of claim 17, wherein the probe or reverse transcriptase-PCR fragment is attached to a solid support. 18. The kit of claim 17, wherein the reverse transcriptase activity and the amplified polymerase activity are found in the same molecule. a) an amplification primer pair capable of annealing to the cDNA of the RNA target, wherein the first amplification primer anneals upstream of the polymorphic sequence position and the second amplification primer anneals downstream of the polymorphic sequence position;
b) a probe comprising a detectable label and a DNA and RNA nucleic acid sequence, wherein the RNA nucleic acid sequence of the probe is completely complementary to the selected site of the cDNA comprising the wild-type DNA sequence at the polymorphic position, a probe that is substantially complementary to a DNA sequence adjacent to a selected portion of the cDNA; And
c) reverse transcriptase activity, amplified polymerase activity, reverse transcriptase-PCR buffer; And a RNase H activity.

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