US2012053069A1PendingUtilityA1

Determination of a Genotype of an Amplification Product at Multiple Allelic Sites

73
Individually held — no corporate assignee on recordPriority: Feb 4, 1998Filed: Nov 13, 2009Published: Mar 1, 2012
Est. expiryFeb 4, 2018(expired)· nominal 20-yr term from priority
Inventors:Kenneth J. LivakFederico Goodsaid
C12Q 1/6858C12Q 1/6818
73
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Claims

Abstract

A method is provided for genotyping a target sequence at 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 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 based on the fluorescence contribution of each fluorescer to the combined fluorescence spectrum.

Claims

exact text as granted — not AI-modified
1 - 54 . (canceled) 
     
     
         55 . A fluorescence spectrum for genotyping a sample of DNA at at least two allelic sites comprising:
 a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA 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 DNA sample 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, 
 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, 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. 
   
     
     
         56 . The spectrum according to  claim 55  wherein the at least two different allelic sites are on a single strand of DNA and amplified by a single set of forward and reverse primers. 
     
     
         57 . The spectrum according to  claim 55  wherein the at least two different allelic sites are on a single strand of DNA and each allelic site is amplified by a different set of forward and reverse primers. 
     
     
         58 . The spectrum according to  claim 55  wherein the at least two different allelic sites are on a separate strands of DNA and each allelic site is amplified by a different set of forward and reverse primers. 
     
     
         59 . The spectrum according to  claim 55  wherein the nucleic acid amplification is performed in the presence of a passive internal standard. 
     
     
         60 . The spectrum according to  claim 59  wherein the passive internal standard is ROX. 
     
     
         61 . The spectrum according to  claim 55  wherein all the oligonucleotide probes include a different fluorescer. 
     
     
         62 . The spectrum according to  claim 55  wherein the forward and reverse primers define amplicons between about 50 and 150 bases in length. 
     
     
         63 . The spectrum according to  claim 55  wherein the one or more sets of forward and reverse primers define amplicons less than about 100 bases in length. 
     
     
         64 . The spectrum according to  claim 55  wherein at least one of the fluorescers is an energy transfer dye. 
     
     
         65 . A fluorescence signature for genotyping a sample of DNA at at least two allelic sites, the signature comprising:
 fluorescence signal contributions of at least three fluorescers to a fluorescence spectrum derived from having performed a nucleic acid amplification on a sample of DNA having at least two different allelic sites using a nucleic acid polymerase having 5′-3′ nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
 each set of allelic oligonucleotide probes is for detecting a different allelic site, 
 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, 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. 
   
     
     
         66 . The fluorescence signature according to  claim 65  wherein the at least two different allelic sites are on a single strand of DNA and amplified by a single set of forward and reverse primers. 
     
     
         67 . The fluorescence signature according to  claim 65  wherein the at least two different allelic sites are on a single strand of DNA and each allelic site is amplified by a different set of forward and reverse primers. 
     
     
         68 . The fluorescence signature according to  claim 65  wherein the at least two different allelic sites are on a separate strands of DNA and each allelic site is amplified by a different set of forward and reverse primers. 
     
     
         69 . The fluorescence signature according to  claim 65  wherein the nucleic acid amplification is performed in the presence of a passive internal standard. 
     
     
         70 . The fluorescence signature according to  claim 69  wherein the passive internal standard is ROX. 
     
     
         71 . The fluorescence signature according to  claim 65  wherein all the oligonucleotide probes include a different fluorescer. 
     
     
         72 . The fluorescence signature according to  claim 65  wherein the forward and reverse primers define amplicons between about 50 and 150 bases in length. 
     
     
         73 . (canceled) 
     
     
         74 . The fluorescence signature according to  claim 65  wherein at least one of the fluorescers is an energy transfer dye. 
     
     
         75 . A library of fluorescence signatures for genotyping a sample of DNA at at least two allelic sites, the library comprising:
 fluorescence signal contributions of at least three fluorescers to a series of fluorescence spectra derived from having performed nucleic acid amplifications on a series of control sequences having known allelic variants at at least two different allelic sites using a nucleic acid polymerase having 5′-3′ nuclease activity and forward and reverse primers capable of hybridizing to the DNA sample in the presence of two or more sets of allelic oligonucleotide probes and amplifying the at least two different allelic sites wherein:
 each set of allelic oligonucleotide probes is for detecting a different allelic site, 
 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, 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. 
   
     
     
         76 - 107 . (canceled)

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