Determination of genotype of an amplification product at multiple allelic sites

Abstract

A method is provided for genotyping a target sequence at least two allelic sites by a 5' nuclease amplification reaction. In one embodiment, the method includes performing a nucleic acid amplification on a target sequence having at least two different allelic sites using a nucleic acid polymerase having 5'-3' nuclease activity and a primer capable of hybridizing to the target sequence in the presence of two or more sets of allelic oligonucleotide probes wherein: each set of allelic oligonucleotide probes is for detecting a different allelic site of the target sequence, each set of allelic oligonucleotide probes includes two or more probes which are complementary to different allelic variants at the allelic site being detected by the set of probes, the allelic site being 5' relative to a sequence to which the primer hybridizes to the target sequence, and at least all but one of the allelic oligonucleotide probes include a different fluorescer than the other probes and a quencher positioned on the probe to quench the fluorescence of the fluorescer; detecting a fluorescence spectrum of the amplification; calculating a fluorescence contribution of each fluorescer to the fluorescence spectrum; and determining a presence or absence of the different allelic variants at the two or more different allelic sites based on the fluorescence contribution of each fluorescer to the combined fluorescence spectrum.

1 - 107 . (canceled) 
     
     
         108 . A method for determining which allelic sites are present in a sample of DNA, the method comprising:
 amplifying the sample of DNA in the presence of: i) a polymerase having 5′ nuclease activity; ii) a forward primer and a reverse primer that hybridize to the sample of DNA, and iii) four oligonucleotide probes that differ from each other by at least one nucleotide base, such that the first and second oligonucleotide probes compete to hybridize to a first allelic site and the third and fourth oligonucleotide probes compete to hybridize to a second allelic site; wherein all of the oligonucleotide probes comprise a different fluorescer, and a quencher that quenches the fluorescence of the fluorescer; and   determining the presence or absence of the first and second allelic sites by detecting a fluorescence spectrum of the amplification reaction resulting from 5′ nuclease digestion of the oligonucleotide probes that hybridized to the sample DNA.   
     
     
         109 . The method according to  claim 108 , wherein the amplification reaction is performed in the presence of a passive internal standard. 
     
     
         110 . The method according to  claim 109 , wherein the passive internal standard is ROX. 
     
     
         111 . The method according to  claim 108 , wherein all the oligonucleotide probes include the same quencher. 
     
     
         112 . The method according to  claim 108 , wherein the amplification reaction is performed in a reaction mixture which includes about 7-9% glycerol, 0.04-0.06% gelatin, 0.005-0.015% TWEEN 20, 25-75 mM tris buffer, pH 8.0, 4-6 mM MgCl 2 , 175-225 uM dATP, 175-225 uM dCTP, 175-225 uM deaza dGTP, 350-450 uM dUTP, 0.045-0.055 U/uL of Taq DNA polymerase, 0.5-0.015 U/uL AmpErase UNG, and 57-63 nM of a Passive Reference. 
     
     
         113 . The method according to  claim 108 , wherein the one or more sets of forward and reverse primers define amplicons between about 50 and 150 bases in length. 
     
     
         114 . The method according to  claim 108 , wherein the % GC of all the probes are at least about 20% and less than about 80%. 
     
     
         115 . The method according to  claim 108 , wherein none of the probes have four or more contiguous guanines. 
     
     
         116 . The method according to  claim 108 , wherein all of the probes have a melting point temperature that is about 3-5° C. greater than an annealing temperature used in the amplification and the primers have a melting point temperature of about 2-4° C. less than the annealing temperature. 
     
     
         117 . The method according to  claim 116 , wherein the annealing temperature is about 60-64° C. 
     
     
         118 . The method according to  claim 116 , wherein all of the probes have a melting point temperature of about 65-67° C. 
     
     
         119 . The method according to  claim 116 , wherein all of the primers have a melting point temperature of about 58-60° C. 
     
     
         120 . The method according to  claim 108 , wherein all of the probes have a melting point temperature about 5-10° C. greater than the melting point temperature of all of the primers. 
     
     
         121 . The method according to  claim 108 , wherein at least one of the probes hybridizes to itself to form a hairpin. 
     
     
         122 . The method according to  claim 108 , wherein the fluorescer on at least one of the probes emits a stronger fluorescence signal when hybridized to a sequence than when not hybridized to a sequence and in a non-hairpin, single stranded form. 
     
     
         123 . The method according to  claim 108 , wherein at least one of the fluorescers is an energy transfer dye. 
     
     
         124 . A kit for identifying in a single 5′ nuclease assay which alleles at multiple different allelic sites are present in a sample of DNA, the kit comprising:
 a first set of oligonucleotide probes comprising first and second oligonucleotide probes for detecting which alleles are present at a first allelic site by single 5′ nuclease assay, the first and second oligonucleotide probes being designed to distinguish between at least two alleles at the first allelic site, which differ from each other by at least one base position, wherein the first set of oligonucleotide probes is digestible by a 5′ to 3′ nuclease, and does not self-hybridize;   a second set of oligonucleotide probes comprising third and fourth oligonucleotide probes for detecting which alleles are present at a second, different allelic site by the single 5′ nuclease assay, the third and fourth oligonucleotide probes being designed to distinguish between at least two alleles at the second allelic site, which differ from each other by at least one base position, wherein the second set of oligonucleotide probes is digestible by a 5′ to 3′ nuclease, and does not self-hybridize; and   a first primer that is capable of hybridizing to a sequence in the first allelic site 3′ relative to where the probes of the first set hybridize to the first allelic site and a second primer that is capable of hybridizing to a sequence in the second allelic site 3′ relative to where the probes of the second set hybridize to the second allelic site;   wherein all of the oligonucleotide probes comprise a different fluorescer and all of the oligonucleotide probes comprise a quencher that quenches the fluorescence of the fluorescer such that the first and second sets of oligonucleotide probes can be used to determine which alleles are present at the first and second allelic sites in the single 5′ nuclease assay.   
     
     
         125 . A kit according to  claim 124 , wherein all of the probes comprise a same quencher, and each quencher and each fluorescer are at opposite ends of each probe. 
     
     
         126 . A kit according to  claim 124 , wherein all of the probes have a melting point temperature that is about 3-5° C. greater than an annealing temperature used in the amplification, and the primers' melting point temperatures are about 2-4° C. less than the annealing temperature. 
     
     
         127 . A kit according to  claim 124 , wherein all of the primers have a melting point temperature of about 58-60° C. 
     
     
         128 . A kit according to  claim 124 , wherein all of the probes have a melting point temperature about 7° C. greater than the melting point temperature of all of the primers. 
     
     
         129 . A kit according to  claim 124 , wherein the % GC of all the probes are at least about 20% and less than about 80%. 
     
     
         130 . A kit according to  claim 124 , wherein none of the probes have four or more contiguous guanines. 
     
     
         131 . A kit according to  claim 124 , wherein all of the probes have a melting point temperature about 65-67° C. 
     
     
         132 . A kit according to  claim 125 , wherein the different fluorescers on the oligonucleotide probes are capable of being excited at the same wavelength. 
     
     
         133 . A kit according to  claim 125 , wherein the oligonucleotide probes are in a non-hairpin, single stranded form when not hybridized to a sequence. 
     
     
         134 . A kit for identifying in a single 5′ nuclease assay which alleles are present in a sample of DNA, the kit comprising:
 first and second oligonucleotide probes for detecting which alleles are present at a first allelic site, wherein the first and second oligonucleotide probes: i) are digestible by a 5′ to 3′ nuclease; ii) do not self hybridize; and iii) differ from each other by at least one nucleotide base;   third and fourth oligonucleotide probes for detecting which alleles are present at a second allelic site, wherein the third and fourth oligonucleotide probes: i) are digestible by a 5′ to 3′ nuclease; ii) do not self hybridize; and iii) differ from each other by at least one nucleotide base;   a first primer that is capable of hybridizing to a sequence of the first allelic site 3′ relative to where the first or second probes hybridize to the first allelic site; and   a second primer that is capable of hybridizing to a sequence of the second allelic site 3′ relative to where the third or fourth probes hybridize to the second allelic site; wherein all of the oligonucleotide probes comprise a different fluorescer and all of the oligonucleotide probes comprise a quencher that quenches the fluorescence of the fluorescer such that the probes can be used to determine which alleles are present at the first and second allelic sites in the single 5′ nuclease assay.