Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

• the present invention broadly concerns methods for detecting specific nucleotide sequences. More particularly, the present invention relates to methodology in which the detection of a signal from an oligonucleotide that is labeled with a molecular energy transfer (MET) pair, including an energy donor and an energy acceptor, indicates the presence of a specific nucleotide sequence in a sample.
• MET molecular energy transfer
• the methodology has enough sensitivity to distinguish a single nucleotide in the context of other nucleotides.
• the invention also concerns detection of nucleic acid polymorphisms, such as single nucleotide polymorphisms (SNPs).
• Certain identifying nucleotide sequences in genes suggest the presence of disease, a susceptibility to disease, or a phenotype. These identifying sequences may represent spontaneous changes in a particular gene (e.g., mutations) of an individual or may represent a difference between forms of a gene (e.g., alleles) that persist in a population. The changes or differences may occur at several nucleotides in a gene or may occur at a single nucleotide. Single-nucleotide changes or differences represent greater challenges for detection systems.
• SNP single-nucleotide polymorphism
• PCR polymerase chain reaction
• a DNA polymerase such as the Klenow fragment of E. coli DNA polymerase I (Saiki et al. (1985), supra) or Thermus aquaticus DNA polymerase (Saiki et al, Science 239: 487-91 (1988)), is used for sequential rounds of template-dependent synthesis of the DNA sequence.
• the DNA Prior to the initiation of each new round, the DNA is denatured and fresh enzyme is added, in the case of the E. coli enzyme. In this manner, exponential amplification of the target sequences is achieved.
• the resultant amplified DNA then can be analyzed readily for the presence of DNA sequence variation, such as the sickle cell mutation, by sequence-specific oligonucleotide hybridization (Saiki et al, Nature 324: 163-66 (1986)), restriction enzyme cleavage (Saiki et al. (1985), supra; Chehab et al, Nature 329: 293-94(1987)), ligation of oligonucleotide pairs (Landegrenet al, Science 241 : 1077-80 (1988)), or ligation amplification. While PCR increased the speed of analysis and reduced the amount of DNA required, it did not change the method of analysis of DNA sequence variation.
• U.S. patent No. 5,639,611 to Wallace et al. disclosed an allele-specific polymerase chain reaction, said to be suitable for detecting the allele responsible for sickle cell anemia.
• Wallace et al. found that amplification proceeds with reduced efficiency when the 3' nucleotide of one of the PCR primers forms a mismatched base-pair with the template.
• the specific primers direct amplification of a specific allele only.
• the formation of an amplified fragment after multiple rounds of amplification, indicates the presence of the allele in the test sample. While Wallace's method represents an improvement over restriction digests and radiolabeled hybridizations, the approach remains laborious and time-consuming.
• the method necessitates performing multiple amplication and detection reactions to identify the genotype of a sample.
• a method for determining the presence in a DNA sample of a first identifying sequence or a second identifying sequence, or both comprises:
• the amplification reaction is PCR
• each of the first and second donor moieties is a fluorophore
• each of the first and second acceptor moieties is a quencher of light emitted by the fluorophore.
• a method for determining the presence in a DNA sample of a first identifying sequence or a second identifying sequence, or both comprises:
• a fourth nucleotide sequence at the 5' end of the third nucleotide sequence wherein the first oligonucleotide is capable of forming a first hairpin containing nucleotides of the second and fourth nucleotide sequences, and the first oligonucleotide emits a first detectable signal if the first hairpin is not formed, wherein the second oligonucleotide contains:
• the present invention also provides a method of directly identifying one or more nucleic acid polymorphisms in a single nucleic acid sample. This improved technique meets two major requirements. First, it permits detection of the nucleic acid polymorphisms without prior separation of unincorporated oligonucleotides. Second, it allows detection of the one or more nucleic acid polymorphisms in a sample directly, by incorporating the labeled oligonucleotide into the amplified nucleic acid sample. The present invention also relates to kits for the identification of one or more nucleic acid polymorphism in a single sample. Such kits may be diagnostic kits where the presence of the nucleic acid polymorphism is correlated with the presence or absence of a disease or disorder.
• FIGURE 1 represents a general schematic of the oligonucleotide primers of the present invention.
• the first oligonucleotide (Ol) comprises a first nucleotide sequence (1) and a second nucleotide sequence (2) at the 5' end of the first nucleotide sequence (hatched).
• the second oligonucleotide (02) comprises a third nucleotide sequence (3) and a fourth nucleotide sequence (4) at the 5' end of the third nucleotide sequence (solid).
• the third oligonucleotide (03) comprises the reverse primer.
• the fourth oligonucleotide (04) comprises a fifth nucleotide sequence (5) (hatched), a sixth nucleotide (6) sequence at the 5' end of the fifth nucleotide sequence, a seventh nucleotide (7) sequence at the 5' end of the sixth nucleotide sequence, and an eighth nucleotide sequence (8) at the 5' end of the seventh nucleotide sequence.
• the fifth nucleotide sequence is identical to the second nucleotide sequence.
• 04 is capable of forming a first hairpin which contains nucleotide sequences six and eight. 04 emits a signal if the hairpin is not formed.
• the fifth oligonucleotide (05) comprises a ninth nucleotide sequence (9) (solid), a tenth nucleotide sequence (10) at the 5' end of the ninth nucleotide sequence, a eleventh nucleotide sequence (1 1) at the 5' end of the tenth nucleotide sequence, and a twelfth nucleotide sequence (12) at the 5' end of the eleventh nucleotide sequence.
• the ninth nucleotide sequence is identical to the fourth nucleotide sequence.
• 05 is capable of forming a hairpin containing nucleotides of the 10 and 12 nucleotide sequences. 05 emits a signal if the hairpin is not formed.
• FIGURE 2 provides a schematic representation of one of the preferred embodiments of the inventive methodology. It employs sequence-specific primers separate from the hairpin- forming oligonucleotides. If a DNA sample contains a target sequence, the sequence-specific forward primer (Ol) will anneal and extend during the first cycle of a polymerase chain reaction, forming an extended first strand. In subsequent cycles, a hairpin- forming oligonucleotide (04) anneals to a 5' tail of the incorporated sequence- specific primer (as in cycle 3) and acts as a primer in formation of a doubly extended first strand.
• sequence-specific forward primer Ol
• a hairpin- forming oligonucleotide anneals to a 5' tail of the incorporated sequence- specific primer (as in cycle 3) and acts as a primer in formation of a doubly extended first strand.
• FIGURE 3 is a schematic illustration of the structure of an oligonucleotide (A) in a hairpin conformation and (B) an extended conformation.
• Element (a) represents a nucleotide sequence that specifically hybridizes to a target sequence;
• (b) and (d) represent nucleotide sequences that hybridize to one another to form a hairpin; and
• (c) represents the sequence that links (b) with (d) and forms the loop of the hairpin.
• Open and closed circles on (b) and (d) represent a pair of molecular energy transfer molecules.
• FIGURE 4 depicts another embodiment of the inventive methodology that utilizes a polymerase chain reaction.
• Hairpin-forming oligonucleotides (a) contain a primer sequence specific for each identifying sequence. If a DNA sample contains a target sequence, the specific primer will anneal and extend during the first cycle of a polymerase chain reaction (forming an extended first strand). In a second cycle, as polymerase copies the extended first strand, it forces the incorporated oligonucleotide out of its hairpin formation and into an extended, light-emitting conformation. Repetitive (n) cycles will increase the magnitude of the signal emitted.
• FIGURE 5A shows the genotyping results of the CYP17 gene for the A-type allele and G-type allele. Both primers differed at their 3' terminal nucleotide. Column 2 represents the fluorescence measurements for FAM, and column 3 represents the fluorescence measurements for SR. Column 1 show the genotype of the CYP17 DNA tested.
• FIGURE 5B illustrates the benefit of using multiplex PCR reaction over a singleplex PCR reaction.
• This figure shows the gentoyping results of the CYP17 gene for the A- type and G-type allele.
• the A-specific primer is labeled with FAM and the G-specific primer is labeled with SR.
• Column 1 a CYP17 DNA sample containing the A-type allele was screened via a singleplex PCR reaction using the A-specific primer.
• Column 2 a CYP17 DNA sample containing the G-type allele was screened via a singleplex PCR reaction using the A-specific primer.
• Column 3 a CYP17 DNA sample containing the A- type allele was screened via a singleplex PCR reaction using the G-specific primer.
• Comparing columns 3 and 4 SR signal is detected in both A-type and G-type DNA samples using the G-specific primer. This result demonstrates the low allelic discrimination using only the G-specific primer in a singleplex PCR reaction. Comparing columns 5 and 6 demonstrates the high allelic discrimination when using both the A-specific and G-specific primers in a multiplex PCR reaction because the allelic there was no signal of the opposite allele generated.
• FIGURE 6A shows the genotyping results of the HER2 gene for the A-type allele and G-type allele. Both primers differed at their 3' terminal nucleotide. The no DNA controls are designated by NDC. Column 1 represents the fluorescence measurements for FAM and column 2 represents the fluorescence measurements for SR. Column 3 show the genotype of the HER2 DNA tested.
• FIGURE 6B shows a graphic representation of the fluorescence results of figure 5A. The results demonstrate the allelic discrimination of the multiplex PCR reaction using both the A-specific and G-specific primers.
• FIGURE 7A shows the genotyping results of the CYP2C8 gene for the C-type allele and T-type allele.
• the 3' terminal nucleotide of the C-specific primer contained the mismatch for detecting the polymorphism.
• the second nucleotide removed from the terminus of the T-specific primer contained the mismatch nucleotide for detecting the polymorphism.
• the no DNA controls are designated by NDC.
• Column 1 represents the fluorescence measurements for FAM and column 2 represents the fluorescence measurements for SR.
• Column 3 show the genotype of the CYP2C8 DNA tested.
• FIGURE 7B shows a graphic representation of the fluorescence results of figure 6A. The results demonstrate the good allelic discrimination of the multiplex PCR reaction using both the C-specific and T-specific primers.
• FIGURE 8A shows the genotyping results of the HTR2C gene for the C-type allele and G-type allele.
• the third nucleotide removed from the terminus of the C-specific and G-specific primer contained the mismatch nucleotide for detecting the polymorphism.
• the no DNA controls are designated by NDC.
• Column 1 represents the fluorescence measurements for FAM and column 2 represents the fluorescence measurements for SR.
• Column 3 show the genotype of the HTR2C DNA tested.
• FIGURE 8B shows a graphic representation of the fluorescence results of figure 7A. The results demonstrate the allelic discrimination of the multiplex PCR reaction using both the C-specific and G-specific primers.
• FIGURE 9A shows the genotyping results of the CCR5 gene for the wild-type gene and the deletion mutant.
• the no DNA controls are designated by NDC.
• Column 1 represents the fluorescence measurements for FAM and column 2 represents the fluorescence measurements for SR.
• Column 3 show the genotype of the CCR5 DNA tested.
• FIGURE 9B shows a graphic representation of the fluorescence results of figure 8A. The results demonstrate the good allelic discrimination of the multiplex PCR reaction using both the wild-type and deletion mutant primers.
• a PCR-based methodology has been discovered for detecting specific nucleotide sequences (identifying sequences), absent the drawbacks of conventional technology.
• the instant invention enables the rapid detection of genetic polymorphisms, such as a SNPs, insertions and deletions, within a target sequence. Elucidation of a polymorphism is accomplished by discerning the presence of one or more identifying sequences via their hybridization to sequence-specific oligonucleotide primers.
• the invention's creative use of fluorescence resonance energy transfer (FRET)-labeled oligonucleotides enables the rapid characterization of these hybridizations.
• FRET fluorescence resonance energy transfer
• an "identifying sequence" refers to a particular nucleotide acid that may represent a variation of a gene that persists in a population, such as an allele, or a spontaneous change in a particular nucleic acid sequence of an individual, such as a mutation.
• the difference or change from the wild type form can encompass a single nucleotide or several nucleotides and typically indicates a particular phenotype, disease or disease-susceptibility .
• a genomic sample from an individual may encompasses two alleles of a particular gene, depending on whether the individual is homozygous or heterozygous for a given gene. Accordingly, a genomic sample can contain a first identifying sequence, a second identifying sequence, both a first and second identifying sequences or no identifying sequences.
• the invention identifies genetic polymorphisms in a DNA sample by simultaneously detecting the presence of a first identifying sequence, a second identifying sequence, or both. To this end, the sample is contacted, in step (A), with first and second oligonucleotides.
• the first and second oligonucleotides may differ from each other in their terminal 3' nucleotide only.
• the oligonucleotides may differ at a nucleotide or at nucleotides other than the 3 ' terminal nucleotide.
• the second, third, fourth, or fifth nucleotides removed from the 3' terminus of each primer may differ individually or in combination.
• the present invention can be used to determine the presence of a variety of identifying sequences simultaneously. For example, where a particular gene has five possible alleles, five oligonucleotides, specific for the corresponding identifying sequences, can be used simultaneously. In this manner, the genotype of the sample can be determined immediately.
• two sequence-specific oligonucleotide primers one specific for a first identifying sequence and one for a second identifying sequence, together with another primer (such as a reverse primer), are used in a PCR mixture containing a genomic DNA template. Primarily, nucleotides toward the 3' end of a primer determine specificity.
• sequence-specific primers may differ from each other in their terminal 3' nucleotide only. Additionally, the primers may differ at a nucleotide or at nucleotides other than the 3' terminal nucleotide. For example, the second, third, fourth, or fifth nucleotides removed from the 3 ' terminus of each primer may differ individually or in combination. When nucleotides other than the 3 ' terminal nucleotide differ, the primer sequences will bridge the polymorphism. Regardless of mismatch location, under appropriate annealing temperature and PCR conditions, each sequence-specific primer only directs amplification using its complementary sequence as a template. Resultant PCR products, representing the target sequences, then are detected by conducting PCR amplification using two FRET-labeled primers, each specific for the PCR product, together with another primer complementary to the PCR products.
• the method comprises the following steps:
• a first oligonucleotide comprising (i) a first nucleotide sequence capable of specifically hybridizing to the first identifying sequence, but unable to specifically hybridize with the second identifying sequence due to one or more nucleotide mismatches, and (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence.
• a second oligonucleotide comprising (i) a third nucleotide sequence capable of specifically hybridizing to the second identifying sequence, but unable to specifically hybridize with the first identifying sequence due to one or more nucleotide mismatches, and (ii) a fourth nucleotide sequence at the 5' end of the first nucleotide sequence.
• step (C) a third oligonucleotide is provided.
• a fourth oligonucleotide comprising (i) a fifth nucleotide sequence, (ii) a sixth nucleotide sequence at the 5' end of the fifth nucleotide sequence, (iii) a seventh nucleotide sequence at the 5' end of the sixth nucleotide sequence, and (iv) an eighth nucleotide sequence at the 5' end of the seventh nucleotide sequence.
• the fifth nucleotide sequence is identical to the second nucleotide sequence, the fourth oligonucleotide is capable of forming a first hairpin which contains nucleotides of the sixth and eighth nucleotide sequences, and the fourth oligonucleotide emits a first detectable signal if the first hairpin is not formed.
• a fifth oligonucleotide comprises (i) a ninth nucleotide sequence, (ii) a tenth nucleotide sequence at the 5' end of the ninth nucleotide sequence, (iii) an eleventh nucleotide sequence at the 5' end of the tenth nucleotide sequence, and (iv) a twelfth nucleotide sequence at the 5' end of the eleventh nucleotide sequence.
• the ninth nucleotide sequence is identical to the fourth nucleotide sequence, the fifth oligonucleotide is capable of forming a second hairpin containing nucleotides of the tenth and twelfth nucleotide sequences, and the fourth oligonucleotide emits a second detectable signal if the second hairpin is not formed.
• step (F) if the first identifying sequence is present in the DNA sample, then (i) the first oligonucleotide anneals with the first identifying sequence, (ii) the 3' end of the first oligonucleotide is extended using the first identifying sequence as a template to form an extended first strand, wherein the first identifying sequence is annealed to the extended first strand, (iii) first identifying sequence is separated from the extended first strand, (iv) the third oligonucleotide is annealed to the extended first strand, (v) the 3' end of the third oligonucleotide is extended using the extended first strand as a template to form an extended second strand, wherein the extended first strand is annealed to the extended second strand, (vi) the extended first strand is separated from the extended second strand, (vii) the fourth oligonucleotide is annealed to the extended second strand, (viii) the 3' end of the fourth
• step (G) if the second identifying sequence is present in the DNA sample, then (i) the second oligonucleotide anneals with the second identifying sequence, (ii) the 3' end of the second oligonucleotide is extended using the second identifying sequence as a template to form an extended third strand, wherein the second identifying sequence is annealed to the extended third strand, (iii) the second identifying sequence is separated from the extended third strand, (iv) the third oligonucleotide is annealed to the extended third strand, (v) the 3' end of the third oligonucleotide is extended using the extended third strand as a template to form an extended fourth strand, wherein the extended third strand is annealed to the extended fourth strand, (vi) the extended third strand is separated from the extended fourth strand, (vii) the fifth oligonucleotide is annealed to the extended fourth strand, (viii) the 3' end of the
• the fourth oligonucleotide emits the first detectable signal only if the first hairpin is not formed, and the fifth oligonucleotide emits the second detectable signal only if the second hairpin is not formed.
• the first detectable signal emitted by the fourth oligonucleotide if the first hairpin is not formed preferably is more intense than a signal emitted by the fourth oligonucleotide if the first hairpin is formed, and the second detectable signal emitted by the fifth oligonucleotide if the second hairpin is not formed is more intense than a signal emitted by the fifth oligonucleotide if the second hairpin is formed.
• the fourth oligonucleotide ideally emits the first detectable signal only if the first hairpin is not formed, and the fifth oligonucleotide emits the second detectable signal only if the second hairpin is not formed.
• the fourth oligonucleotide contains a first molecular energy transfer pair including a first energy donor moiety that is capable of emitting a first energy, and a first energy acceptor moiety that is capable of absorbing an amount of the emitted first energy.
• the first donor moiety is attached to a nucleotide of the sixth nucleotide sequence and the first acceptor moiety is attached to a nucleotide of the eighth nucleotide sequence, or the first acceptor moiety is attached to a nucleotide of the sixth nucleotide sequence and the first donor moiety is attached to a nucleotide of the eighth nucleotide sequence, and the first acceptor moiety absorbs the amount of the emitted first energy only if the first hairpin is formed.
• the fifth oligonucleotide further contains a second molecular energy transfer pair including a second energy donor moiety that is capable of emitting a second energy, and a second energy acceptor moiety that is capable of absorbing an amount of the emitted second energy.
• the second donor moiety is attached to a nucleotide of the tenth nucleotide sequence and the second acceptor moiety is attached to a nucleotide of the twelfth nucleotide sequence, or the second acceptor moiety is attached to a nucleotide of the tenth nucleotide sequence and the second donor moiety is attached to a nucleotide of the twelfth nucleotide sequence, and the second acceptor moiety absorbs the amount of the emitted second energy only if the second hairpin is formed.
• Each of the first and second donor moieties can be a fluorophore, and each of the first and second acceptor moieties is a quencher of light emitted by the fluorophore.
• the preferred first and second acceptor moieties are DABSYL, while the preferred first donor moiety is fluorescein and the preferred second acceptor moiety is sulfarhodamine, or vice versa.
• the amplification reaction is a polymerase chain reaction, e.g., a triamplification, a nucleic acid sequence-based amplification, a strand displacement amplification, a cascade rolling circle amplification, or an amplification refractory mutation system.
• the amplification reaction may be conducted in situ.
• Step (F)(xii) ideally comprises (a) separating the doubly extended first strand from the doubly extended second strand, (b) annealing the third oligonucleotide to the doubly extended first strand, and annealing the fourth oligonucleotide to the doubly extended second strand, (c) extending the 3' end of the third oligonucleotide using the doubly extended first strand as a template to form another doubly extended second strand, wherein the doubly extended first strand is annealed to the other doubly extended second strand, and extending the 3' end of the fourth oligonucleotide using the doubly extended second strand as a template to form another doubly extended first strand, wherein the doubly extended second strand is annealed to the other doubly extended first strand, and (d) repeating (a), (b), and (c) for a finite number of times, wherein, in (a), the doubly extended first and second strand
• Step (G)(xii) ideally comprises separating the doubly extended third strand from the doubly extended fourth strand, (b) annealing the third oligonucleotide to the doubly extended third strand, and annealing the fifth oligonucleotide to the doubly extended fourth strand, (c) extending the 3' end of the third oligonucleotide using the doubly extended third strand as a template to form another doubly extended fourth strand, wherein the doubly extended third strand is annealed to the other doubly extended fourth strand, and extending the 3' end of the fifth oligonucleotide using the doubly extended fourth strand as a template to form another doubly extended third strand, wherein the doubly extended fourth strand is annealed to the other doubly extended third strand, and (d) repeating (a), (b), and (c) for a finite number of times, wherein, in (a), the doubly extended third and fourth strands respectively are
• the molecular energy transfer (MET) phenomenon is a process by which energy is passed between a donor molecule and an acceptor molecule.
• Fluorescence resonance energy transfer (FRET) which involves at least one fluorophore, is a form of MET.
• a fluorophore is a compound that absorbs light at one wavelength, and emits light at different wavelength.
• a spectrofluorimeter is used to simultaneously emit light which excites the fluorophore, and detect light emitted by the fluorophore.
• FRET the fluorophore is a donor molecule which absorbs photons, and subsequently transfers this energy to an acceptor molecule.
• Donor and acceptor molecules that engage in MET or FRET are termed "MET pairs" and "FRET pairs,” respectively. F ⁇ rster, Z. Naturforsch A4: 321-27 (1994); Clegg, Methods In Enzymology 21 1 : 353-88 (1992).
• excitation of the first fluorophore causes it to emit light that is absorbed by the second fluorophore, which in turn causes the second fluorophore to emit light.
• the fluorescence of the first fluorophore is quenched, while the fluorescence of the second fluorophore is enhanced. If the energy of the first fluorophore is transferred to a compound that is not a fluorophore, however, the fluorescence of the first fluorophore is quenched without subsequent emission of light by the non-fluorophore.
• the FRET phenomenon has been exploited to detect nucleic acids.
• One of these methods is disclosed in U.S. patent No. 5,866,366, the entire contents of which are herein incorporated by reference.
• the '366 patent relates a FRET-labeled hairpin oligonucleotide which is used as a probe in polymerase chain reaction (PCR) methods to detect target nucleic acid sequences.
• This oligonucleotide contains an energy donor and an energy acceptor constituting a FRET pair.
• the donor and acceptor are respectively situated on first and second nucleotide sequences of the oligonucleotide. These two nucleotide sequences are complementary to each other, and are therefore able to form a hairpin in the oligonucleotide.
• the donor and acceptor are in close proximity. In this spatial arrangement, the acceptor absorbs the emission from the donor, and thereby quenches the signal from the donor. However, if the nucleotide sequences are not annealed to each other, then the donor and acceptor are separated, the acceptor can no longer absorb the emission from the donor, and the signal from the donor is not quenched.
• the hairpin unfolds, resulting in the separation of the donor from the acceptor, and the consequent emission of an observable signal.
• the hairpin remains, and the emission from the donor is quenched by the acceptor. Detection of a signal after PCR therefore indicates the presence of the target.
• the concentration of the first oligonucleotide in the reaction mixture in which (F)(i) is conducted is less than 50 nM, 25 nM, or 5 nM, is between 5 and 50 nM or between 20 and 30 nM, or is about 25 nM.
• the concentration of the fourth oligonucleotide in the reaction mixture in which (F)(vii) is conducted is preferably less than 500 nM, 250 nM, or 50 nM, is between 50 and 500 nM or between 200 and 300 nM, or is about 250 nM.
• the concentration of the fourth oligonucleotide in the reaction mixture in which (F)(vii) is conducted is at least five times, ten times, twenty times, thirty times, from five to thirty times, from ten to twenty times, or about ten times the concentration of the first oligonucleotide in the reaction mixture in which (F)(i) is conducted.
• the concentration of the second oligonucleotide in the reaction mixture in which (F)(i) is conducted is less than 50 nM, 25 nM, 5 nM, is between 5 and 50 nM or between 20 and 30 nM, or is about 25 nM.
• the concentration of the fifth oligonucleotide in the reaction mixture in which (F)(vii) is conducted is less than 500 nM, 250 nM, 50 nM, is between 50 and 500 nM or between 200 and 300 nM, or is about 250 nM.
• the concentration of the fifth oligonucleotide in the reaction mixture in which (F)(vii) is conducted is at least five times, ten times, twenty times, thirty times, from five to thirty times, from ten to twenty times, or about ten times the concentration of the second oligonucleotide in the reaction mixture in which (F)(i) is conducted.
• the present invention provides a method for the direct identification of a nucleic acid polymorphism, where the detection may be performed without opening the reaction tube.
• the "closed-tube" format reduces greatly the possibility of carryover contamination with amplification products that has slowed the acceptance of PCR in many applications.
• the closed-tube method also provides for high throughput of samples and may be totally automated.
• the nucleic acids in the sample may be purified or unpurified.
• the oligonucleotides of the invention are used in situ amplification reactions, performed on samples of fresh or preserved tissues or cells.
• in situ reactions it is advantageous to use methods that allow for the accurate and sensitive detection of the target directly after the amplification step.
• conventional in situ PCR requires, in paraffin embedded tissue, detection by a hybridization step, as the DNA repair mechanism invariably present in tissue samples from, e.g., CNS, lymph nodes, and spleen, precludes detection by direct incorporation of a reporter nucleotide during the PCR step.
• the energy emitted by the donor moiety e.g. , when a quencher is the acceptor moiety
• the acceptor moiety e.g., when a fluorophore or chromophore is the acceptor moiety
• the methods of the invention can be used quantitatively to determine the existence of a nucleic acid polymorphism, number of chromosomes, or amount of DNA or RNA, containing the preselected target sequence.
• the first oligonucleotide contains (i) a first nucleotide sequence, (ii) a second nucleotide sequence at the 5' end of the first nucleotide sequence, (iii) a third nucleotide sequence at the 5' end of the second nucleotide sequence, and (iv) a fourth nucleotide sequence at the 5' end of the third nucleotide sequence.
• the first oligonucleotide would form a first hairpin, containing nucleotides of the second and fourth nucleotide sequences ( Figure 3A).
• the first oligonucleotide emits a first detectable signal if the first hairpin is not formed ( Figure 3B).
• the second oligonucleotide contains (i) a fifth nucleotide sequence, (ii) a sixth nucleotide sequence at the 5' end of the fifth nucleotide sequence, (iii) a seventh nucleotide sequence at the 5' end of the sixth nucleotide sequence, and (iv) an eighth nucleotide sequence at the 5' end of the seventh nucleotide sequence.
• the second oligonucleotide is capable of forming a second hairpin containing nucleotides of the sixth and eighth nucleotide sequences, and the second oligonucleotide emits a second detectable signal if the second hairpin is not formed.
• the first oligonucleotide is incorporated into a double-stranded nucleic acid, by means of a polymerase, if the first identifying sequence is present in the sample, thereby preventing the first hairpin from forming ( Figure 4, through 2 cycles).
• a polymerase effects the incorporation of the second oligonucleotide into a double-stranded nucleic acid, if the second identifying sequence is present in the sample, thereby preventing the second hairpin from forming. If both the first and second identifying sequences are present in the sample, each of the first and second oligonucleotides is incorporated into a double-stranded nucleic acid, precluding formation of the first and second hairpins, respectively.
• step (C) an amplification reaction is conducted ( Figure 4, through n cycles).
• the result is (i) incorporation of the first oligonucleotide into a first amplification product, if the first identifying sequence is present in the sample, (ii) incorporation of the second oligonucleotide into a second amplification product, if the second identifying sequence is present in the sample, or (iii) incorporation of the first oligonucleotide into the first amplification product and the second oligonucleotide into the second amplification product, if both identifying sequences are present in the sample.
• step (D) a determination is made as to whether the first identifying sequence is present in the sample (i.e. , if the first signal is detected), whether the second identifying sequence is present (if the second signal is detected), or whether both identifying sequences are present in the sample (both signals are detected).
• the first oligonucleotide emits the first detectable signal only if the first hairpin is not formed, and the second oligonucleotide emits the second detectable signal only if the second hairpin is not formed.
• the first detectable signal, emitted by the first oligonucleotide if the first hairpin is not formed preferably is more intense than a signal emitted by the first oligonucleotide if the first hairpin is formed
• the second detectable signal, emitted by the second oligonucleotide if the second hairpin is not formed is more intense than a signal emitted by the second oligonucleotide if the second hairpin is formed.
• the first oligonucleotide further contains a first molecular energy transfer pair which includes a first energy donor moiety that is capable of emitting a first energy, and a first energy acceptor moiety that is capable of absorbing an amount of the emitted first energy.
• the first donor moiety is attached to a nucleotide of the second nucleotide sequence and the first acceptor moiety is attached to a nucleotide of the fourth nucleotide sequence, or the first acceptor moiety is attached to a nucleotide of the second nucleotide sequence and the first donor moiety is attached to a nucleotide of the fourth nucleotide sequence, and the first acceptor moiety absorbs the amount of the emitted first energy only if the first hairpin is formed.
• the second oligonucleotide further contains a second molecular energy transfer pair including a second energy donor moiety that is capable of emitting a second energy, and a second energy acceptor moiety that is capable of absorbing an amount of the emitted second energy.
• the second donor moiety is attached to a nucleotide of the sixth nucleotide sequence and the second acceptor moiety is attached to a nucleotide of the eighth nucleotide sequence, or the second acceptor moiety is attached to a nucleotide of the sixth nucleotide sequence and the second donor moiety is attached to a nucleotide of the eighth nucleotide sequence, and the second acceptor moiety absorbs the amount of the emitted second energy only if the second hairpin is formed.
• each of the first and second donor moieties is a fluorophore, and that each of the first and second acceptor moieties is a quencher of light emitted by the fluorophore.
• Illustrative of the first and second acceptor moieties is DABSYL, while the first donor moiety can be fluorescein and the second acceptor moiety can be sulfarhodamine; alternatively, the first donor moiety is sulfarhodamine and the second acceptor moiety is fluorescein.
• the amplification reaction ideally is a polymerase chain reaction, such as a triamplification, a nucleic acid sequence-based amplification, a strand displacement amplification, a cascade rolling circle amplification, or an amplification refractory mutation system.
• the amplification reaction may be conducted in situ.
• nucleic acids that are "complementary" can be perfectly or imperfectly complementary, as long as the desired property resulting from the complementarity is not lost, e.g., ability to hybridize.
• kits for determining the presence of a first identifying sequence or a second identifying sequence or both comprise first to fifth oligonucleotides, in one or more containers.
• the kit can further comprise additional components for carrying out the amplification reactions of the invention.
• the target nucleic acid sequence being amplified is one implicated in disease or disorder, the kits can be used for diagnosis or prognosis.
• a kit is provided that comprises, in one or more containers, forward and reverse primers of the invention for carrying out detection and amplification of the nucleic acid polymorphism, and optionally, a DNA polymerase or two DNA polymerases respectively with and without exonuclease activity.
• a kit for triamplification can further comprise, in one or more containers, a blocking oligonucleotide, and optionally DNA ligase.
• Oligonucleotides in containers can be in any form, e.g. , lyophilized, or in solution (e.g. , a distilled water or buffered solution), etc.
• oligonucleotides ready for use in the same amplification reaction can be combined in a single container or can be in separate containers.
• Multiplex kits are also provided, containing more than one pair of amplification (forward and reverse) primers, wherein the signal being detected from each amplified product is of a different wavelength, e.g., wherein the donor moiety of each primer pair fluoresces at a different wavelength. Such multiplex kits contain at least two such pairs of primers.
• a kit comprises, in one or more containers, a pair of primers preferably in the range of 10-100 or 10-80 nucleotides, and more preferably, in the range of 20-40 nucleotides, that are capable of priming amplification.
• primers can initiate amplification in a variety of amplification reactions, including, but not limited to, PCR (see e.g. , Innis et al. , 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.), competitive PCR, competitive reverse-transcriptase PCR (Clementi et al. , 1994, Genet. Anal. Tech. Appl. ll(l):l-6; Siebert et al. , 1992, Nature 359:557-558), triamplification, NASBA and strand displacement.
• PCR see e.g. , Innis et al. , 1990, PCR Protocols, Academic Press, Inc., San Diego, Calif.
• competitive PCR competitive reverse-
• a pair of primers consisting of a forward primer and a reverse primer, for use in PCR or strand displacement amplification, consists of primers that are each complementary with a different strand of two complementary nucleic acid strands, such that when an extension product of one primer in the direction of the other primer is generated by a nucleic acid polymerase, that extension product can serve as a template for the synthesis of the extension product of the other primer.
• a pair of primers consisting of a forward primer and a reverse primer, for use in triamplification, consists of primers that are each complementary with a different strand of two complementary nucleic acid strands, such that when an extension-ligation product of one primer in the direction of the other primer is generated by a nucleic acid polymerase and a nucleic acid ligase, that extension-ligation product can serve as a template for the synthesis of the extension-ligation product of the other primer.
• the amplified product in these instances is that content of a nucleic acid in the sample between and including the primer sequences.
• a kit for determining the presence of a first identifying sequence or a second identifying sequence or both comprising in one or more containers (a) oligonucleotide primers, one or both of which are hairpin primers labeled with fluorescent and quenching moieties that can perform MET; and optionally: (b) a control DNA target sequence; (c) an optimized buffer for amplification; (d) appropriate enzymes for the method of amplification contemplated, e.g., a DNA polymerase for PCR or triamplification or SDA, or a reverse transcriptase for NASBA; and (e) a set of directions for carrying out the amplification.
• Such directions can describe, for example, the optimal conditions, e.g.
• the kit provides (f) means for stimulating and detecting fluorescent light emissions, e.g. , a fluorescence plate reader or a combination thermocycler-plate-reader to perform the analysis.
• means for stimulating and detecting fluorescent light emissions e.g. , a fluorescence plate reader or a combination thermocycler-plate-reader to perform the analysis.
• a kit for triamplification comprises forward and reverse extending primers and a blocking oligonucleotide.
• Either the forward or reverse primer is labeled with one moiety of a pair of MET moieties, and the blocking oligonucleotide is labeled with the other MET moiety of the pair.
• kits comprises, in one or more containers: (a) a first oligonucleotide; (b) a second oligonucleotide, wherein said first and second oligonucleotides are linear primers for use in a triamplification reaction; (c) a third oligonucleotide that is a blocking oligonucleotide that comprises a sequence complementary and hybridizable to a sequence of said first oligonucleotide, said first and third oligonucleotides being labeled with a first and second moiety, respectively, that are members of a molecular energy transfer pair consisting of a donor moiety and an acceptor moiety, such that when said first and third oligonucleotides are hybridized to each other and the donor moiety is excited and emits energy, the acceptor moiety absorbs energy emitted by the donor moiety; and (d) in a separate container, a nucleic acid ligase.
• kits comprises in a container a universal hairpin optionally also comprising a second container containing cyanogen bromide or a nucleic acid ligase (e.g. , DNA ligase, for example, T4 DNA ligase).
• a nucleic acid ligase e.g. , DNA ligase, for example, T4 DNA ligase
• a kit for carrying out a reaction such as that shown in FIG. 2 comprises in one or more containers: (a) a first and second oligonucleotide; (b) a third oligonucleotide, wherein the first and second oligonucleotides are forward primers and the third oligonucleotide is a reverse primer for DNA synthesis in an amplification reaction to identify a nucleic acid polymorphism, and wherein said first and second oligonucleotides comprise (i) a 5' sequence that is not complementary to a preselected target sequence in said nucleic acid sequence, and (ii) a 3' sequence that is complementary to said preselected target sequence and may comprise one or more mismatch nucleotides; and (c) a fourth oligonucleotide that comprises in 5' to 3' order (i) a first nucleotide sequence of 6-30 nucleotides, wherein a nucleotide within said first nucleotide sequence is
• kits of the invention comprises in one or more containers: (a) a first oligonucleotide; (b) a second oligonucleotide, said first and second oligonucleotide being hybridizable to each other; said first oligonucleotide being labeled with a donor moiety said second oligonucleotide being labeled with an acceptor moiety, said donor and acceptor moieties being a molecular energy transfer pair, wherein the donor moiety emits energy of one or more particular wavelengths when excited, and the acceptor moiety absorbs energy at one or more particular wavelengths emitted by the donor moiety; and (c) in a separate container, a nucleic acid ligase.
• the approach of the present invention presents a powerful tool for genetic disease diagnosis, carrier screening, HLA typing, human gene mapping, forensics, and paternity testing, inter alia.
• the invention is further described by reference to the following example, which is set forth by way of illustration only. None in the following examples should be taken as a limitation upon the overall spirit and scope of the present invention.
• FIG. 1 A schematic representation of the oligonucleotide primers of the present invention are shown in Figure 1.
• oligonucleotide primers of the present invention will be referred as the primers listed below:
• forward primer This is a sequence specific primer that contains one or more mismatched nucleotides for the detection of one or more polymorphisms and is capable of binding to the FAM universal primer;
• forward primer This is the second sequence specific primer that contains one or more mismatched nucleotides for detection one or more polymorphisms. This primer is capable of binding to the SR universal primer;
• SR universal primer 05 will herein be referred to as the "SR universal primer”.
• Table 1 illustrates representative examples of both forward and reverse primers.
• the sequence specific forward primer pairs in Table 1 differ at their terminal 3' nucleotide.
• Table 2 illustrates representative examples of both forward and reverse primers, as well as the gene target sequence containing the polymorphism.
• the universal FRET primers used in the following examples comprise the following sequences:
• sequences capable of forming the hairpin are underlined in both universal primers and the italicized T shows where the DABSYL quencher is tethered to a base.
• Amplification reactions were assembled in standard 96-well polypropylene PCR plates reactions, which can be read directly on an a Victor II fluorescence plate reader (Wallac). Alternatively, reactions can take place in tubes, and then be transferred to plates for fluorescence measurements.
• the PCR amplification reaction mixture is listed below.
• Oligonucleotide 4-FAM (Seq ID No:l) 250 nM
• Oligonucleotide 5-SR (Seq ID No: 2) 250 nM
• Tris HC1 (pH 8.3) 100 M dNTPs 200 ⁇ M each rTaq Polymerase (Shuzo Co, Japan) 0.5 units
• PCR Reaction The UltraPIates were sealed with cyclesealer (Robbins Scientific), placed onto the thermocycler block (Perkin-Elmer 9700) and preheated to 94°C. After heating at 94°C for 5 minutes, 35 cycles of amplification (10 seconds at 94°C, 20 seconds at 55°C, 40 seconds at 72°C) followed.
• Fluorescence measurement Following the reaction, the plate was placed in a black support to prevent cross-talk, and fluorescence intensity was measured with green and red filters. Several instruments were found to be adequate for the fluorescence measurement of the samples, two fluorescent plate readers and two digital cameras.
• Amplification Reaction Samples of human genomic DNA were analyzed for the presence of *2 allele in the CYP 17 gene. In particular, the DNA samples were analyzed for the presence of either the A-type or G-type allele.
• the unlabeled forward primer specific for the A-type allele comprised the sequence 5'-gaaggtgaccaagttcatgctGCCACAGCTCTTCTACTCCACT (SEQ ID No: 12), where the sequence identical to the 3 '-portion of the FAM labeled primer is shown in lower case.
• the unlabeled forward primer specific for the G-type allele comprised the sequence 5'-gaaggtcggagtcaacggattGCCACAGCTCTTCTACTCCACC, where the sequence identical to the 3 '-portion of the SR labeled primer is shown in lower case.
• the nucleotides directed to identifying the polymorphism are shown in bold.
• the reverse primer used in the reaction comprised the sequence GGCACCAGGCCACCTTCTCTT (SEQ ID No: 14).
• the universal FRET primers used were identical to those described in Example 1.
• Reaction 1 Singleplex: The amplification reaction mixture using human genomic DNA was the same as that described in Example 1, with the following differences. This reaction only utilized the primer specific for the A-type allele, the FAM labeled hairpin primer, and the reverse primer.
• Reaction 2 Singleplex: The amplification reaction mixture using human genomic DNA was the same as that described in Example 1, with the following differences. This reaction only utilized the primer specific for the G-type allele, the SR-labeled hairpin primer, and the reverse primer.
• Reaction 3 (Multiplex): The amplification reaction mixture using human genomic DNA was the same as that described in Example 1. This reaction used both of the forward primers, i.e., the primer specific for the G-type allele and the primer specific for the A-type allele, the SR- labeled hairpin primer, the FAM-labeled hairpin primer, and the reverse primer. This reaction is exemplary of multiplex PCR amplification because both allele specific primers are present in the same reaction. 3. PCR Reaction
• the three sets of reactions each included three 'no DNA controls' (NDC).
• NDC 'no DNA controls'
• the mixtures were preheated at 94°C for 3 min and subjected to thermocycling (PCR) for 35 cycles of 10 sec at 94°C, 20 sec at 55°C, and 40 sec at 72°C.
• Figures 5 A and 5B show that the A-specific primer had produced FAM signal on both A-type and G-type DNA (1 and 2), and the G-specific primer produced SR signal with both A-type and G-type DNA (3 and 4). Hence, when these primers were taken separately, the allelic discrimination was low and not sufficient to distinguish between the types of DNA. When both the A-specific and G- specific primers, however, were present simultaneously (reaction 3, multiplex PCR), (5 and 6).
• Example 3 Multiplex allele-specific PCR for detecting a SNP in the HER2 gene 1.
• Amplification Reaction Samples of genomic DNA were analyzed for the presence of a SNP in the HER2 gene. In particular, the DNA samples were analyzed for the presence of either the A-type or G-type allele.
• the primers and target sequence are summarized in Table 2(A).
• the unlabeled forward primer specific for the A-type allele comprised the sequence 5'-gaaggtgaccaagttcatgctGCC ACCACCGCAGAG T (SEQ ID No: XX), where the sequence identical to the 3 '-portion of the FAM labeled primer is shown in lower case and the nucleotide directed to identifying the polymorphism is shown in bold.
• the unlabeled forward primer specific for the G-type allele comprised the sequence 5'-gaaggtcggagtcaacggattGCCAACCACCGCAGAGAC (Seq ID No: XX), where the sequence identical to the 3 '-portion of the SR labeled primer is shown in lower case, and the nucleotide directed to identifying the polymorphism is shown in bold.
• the reverse primer used in the reaction comprised the sequence TCAATCCCTGACCCTGGCTT (SEQ ID No: XX).
• the universal FRET primers used were identical to those described in Example 1.
• the amplification reaction mixture using genomic DNA was the same as that described in Example 1. This reaction used both of the forward primers, i.e., the primer specific for the G-type allele and the primer specific for the A-type allele, the SR-labeled hairpin primer, the FAM-labeled hairpin primer, and the reverse primer.
• PCR reactions each included four 'no DNA controls' (NDC).
• NDC 'no DNA controls'
• the mixtures were preheated at 94°C for 3 min and subjected to thermocycling (PCR) for 35 cycles of 10 sec at 94°C, 20 sec at 55°C, and 40 sec at 72°C.
• Example 4 Multiplex allele-specific PCR for detecting a SNP in CYP2C8 gene by bridging the polymorphism
• Amplification Reaction Samples of genomic DNA were analyzed for the presence of a SNP in the CYP2C8 gene. In particular, the DNA samples were analyzed for the presence of either the C-type or T-type allele.
• the primers used in this reaction differ from each other. The primer specific for the T-type allele will bridge the polymorphism because a nucleotide other than its 3' terminal nucleotide differs.
• the primers and target sequence are summarized in Table 2(B).
• the unlabeled forward primer specific for the C-type allele comprised the sequence 5'-gaaggtgaccaagttcatgctGTTGCAGGTGATAGCAGATCG (SEQ ID No: XX), where the sequence identical to the 3 '-portion of the FAM labeled primer is shown in lower case, and the nucleotide directed to identifying the polymorphism is shown in bold.
• the unlabeled forward primer specific for the T-type allele comprised the sequence 5'-gaaggtcggagtcaacggattGTTGCAGGTGATAGCAGATAG (Seq ID No: XX), where the sequence identical to the 3 '-portion of the SR labeled primer is shown in lower case, and the nucleotide directed to identifying the polymorphism is shown in bold. With this primer, the second nucleotide removed from the 3' terminus of the primer differs rather than the 3' terminal nucleotide.
• the reverse primer used in the reaction comprised the sequence TGCTTCATCCCTGTCTGAAGAAT (SEQ ID No: XX).
• the universal FRET primers used were identical to those described in Example 1.
• the amplification reaction mixture using genomic DNA was the same as that described in Example 1. This reaction used both of the forward primers, i.e., the primer specific for the c-type allele and the primer specific for the t-type allele, the SR-labeled hairpin primer, the FAM-labeled hairpin primer, and the reverse primer.
• PCR reactions each included four 'no DNA controls' (NDC).
• NDC 'no DNA controls'
• the mixtures were preheated at 94°C for 3 min and subjected to thermocycling (PCR) for 35 cycles of 10 sec at 94°C, 20 sec at 55°C, and 40 sec at 72°C. 4.
• PCR thermocycling
• Example 5 Multiplex allele-specific PCR for detecting a SNP in HTR2C gene by bridging the polymorphism
• Amplification Reaction Samples of genomic DNA were analyzed for the presence of a SNP in the HTR2C gene. In particular, the DNA samples were analyzed for the presence of either the C-type or G-type allele. The primers used in this reaction will bridge the polymorphism because the 3 nucleotide removed from the 3' terminal nucleotide differs.
• the primers and target sequence are summarized in Table 2(C).
• the unlabeled forward primer specific for the C-type allele comprised the sequence 5'-gaaggtgaccaagttcatgctGGGCTCACAGAAATATCAGAT (SEQ ID No: XX), where the sequence identical to the 3 '-portion of the FAM labeled primer is shown in lower case and the nucleotide directed to identifying the polymorphism is shown in bold.
• the unlabeled forward primer specific for the T-type allele comprised the sequence 5'-gaaggtcggagtcaacggattGGGCTCACAGAAATATCACAT (Seq ID No: XX), where the sequence identical to the 3 '-portion of the SR labeled primer is shown in lower case and the nucleotide directed to identifying the polymorphism is shown in bold. With this primer, the second nucleotide removed from the 3' terminus of the primer differs rather than the 3' terminal nucleotide.
• the reverse primer used in the reaction comprised the sequence TGCACCTAATTGGCCTATTGGTTT (SEQ ID No: XX).
• the universal FRET primers used were identical to those described in Example 1. 2.
• the amplification reaction mixture using genomic DNA was the same as that described in Example 1. This reaction used both of the forward primers, i.e., the primer specific for the G-type allele and the primer specific for the C-type allele, the SR-labeled hairpin primer, the FAM-labeled hairpin primer, and the reverse primer.
• PCR reactions each included four 'no DNA controls' (NDC).
• NDC 'no DNA controls'
• the mixtures were preheated at 94°C for 3 min and subjected to thermocycling (PCR) for 35 cycles of 10 sec at 94°C, 20 sec at 55°C, and 40 sec at 72°C.
• Example 6 Multiplex allele-specific PCR for detecting a deletion in the CCR5 gene 1.
• Amplification Reaction Samples of genomic DNA were analyzed for the presence of a deletion in the CCR5 gene. In particular, the DNA samples were analyzed for the presence of absence of the gene deletion.
• the primers and target sequence are summarized in Table 2(D).
• the unlabeled forward primer specific for the wild- type allele comprised the sequence 5'-gaaggtgaccaagttcatgctCTCATTTTCCATACAGTCA (SEQ ID No: XX), where the sequence identical to the 3 '-portion of the FAM labeled primer is shown in lower case and the nucleotide directed to identifying the wild-type allele is shown in bold.
• the unlabeled forward primer specific for the mutant allele comprised the sequence 5'-gaaggtcggagtcaacggattgcagctctcattttccatacatta (Seq ID No: XX), where the sequence identical to the 3 '-portion of the SR labeled primer is shown in lower case.
• the reverse primer used in the reaction comprised the sequence ACCAGCCCCAAGATGACTATCTT (SEQ ID No: XX).
• the universal FRET primers used were identical to those described in Example 1.
• the amplification reaction mixture using genomic DNA was the same as that described in Example 1. This reaction used both of the forward primers, i.e., the primer specific for the wild type-allele and the primer specific for the mutant-allele, the SR-labeled hairpin primer, the FAM-labeled hairpin primer, and the reverse primer.
• PCR reactions each included four 'no DNA controls' (NDC).
• NDC 'no DNA controls'
• the mixtures were preheated at 94°C for 3 min and subjected to thermocycling (PCR) for 35 cycles of 10 sec at 94°C, 20 sec at 55°C, and 40 sec at 72°C.

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