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US 2020/0263247 A1Published application
Methods of determining the presence or absence of a plurality of target polynucleotides in a sample
AssigneesAffymetrix, Inc., The United States of America as represented by the Secretary of Agriculture
InventorsJohn D. CURRY, Heather KOSHINSKY, Amanda . LINDHOLM-PERRY, Richard M. THALLMAN
Life of this patent
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Abstract
A method for detecting the presence or absence of a target polynucleotide in a sample is described.
Claims19
A method of determining the presence or absence of a target polynucleotide in a sample, the method comprising:
- a) combining
- i) a sample comprising one or more target polynucleotides, the target polynucleotide comprising a first target sequence and a second target sequence;
- ii) a first complementary polynucleotide comprising a first complementary sequence, wherein the first complementary sequence is complementary to the first target sequence of the target polynucleotide;
- and
- iii) a second complementary polynucleotide comprising a second complementary sequence, wherein the second complementary sequence is complementary to the second target sequence of the target polynucleotide;
- b) incubating the first and second complementary polynucleotides to the target polynucleotide under conditions that allow hybridization of complementary sequences;
- c) if the first complementary polynucleotide and the second complementary polynucleotide are hybridized to the same target polynucleotide, then joining the first complementary polynucleotide to the second complementary polynucleotide, to form a first product polynucleotide;
- and
- d) detecting the presence or absence of the product polynucleotide.
6 more elements
A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:
- a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;
- wherein each of the plurality of sets of complementary polynucleotides comprises:
- (i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide,
- (ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide;
- and
- (iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,
- wherein the allele-specific barcodes are not complementary to the first target sequence:
- b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;
- c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides;
- and
- d) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target polynucleotide;
- and
- e) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7.
10 more elements
A method of determining the allele frequency of one or more target polynucleotides of a plurality of target polynucleotides in a sample, the method comprising:
- a) combining a sample comprising one or more of the plurality of target polynucleotides with a plurality of sets of complementary polynucleotides, said one or more of the plurality of target polynucleotides suspected to have a site of a single nucleotide polymorphism (SNP) containing a polymorphic nucleotide;
- wherein each of the plurality of sets of complementary polynucleotides comprises;
- (i) a first complementary polynucleotide comprising a complementary sequence to a first target sequence of a target polynucleotide, a first allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a first polymorphic nucleotide;
- (ii) a second complementary polynucleotide comprising a complementary sequence to the first target sequence, a second allele-specific barcode, and a site of a single nucleotide polymorphism (SNP) containing a second polymorphic nucleotide;
- and
- (iii) a third complementary polynucleotide comprising a complementary sequence to a second target sequence of the target polynucleotide,
- wherein the allele-specific barcodes are not complementary to the first target sequence and are 5 to 15 nucleotides in length;
- b) incubating the plurality of sets of complementary polynucleotides with the plurality of target polynucleotides under conditions that allow hybridization of complementary sequences;
- c) joining a pair of the first and third complementary polynucleotides and/or joining a pair of the second and third complementary polynucleotides by a ligation reaction when both complementary polynucleotides of each pair are hybridized to the target polynucleotide to form one or more product polynucleotides;
- d) amplifying the one or more product polynucleotides, wherein each of the first, second, and third complementary polynucleotides comprises a sequence complementary to an amplification primer bound by a primer for the amplifying, wherein at least one of the sequences complementary to an amplification primer comprises a sample-specific tag sequence corresponding to the identity of the sample;
- and
- e) detecting the presence of one or more product polynucleotides to determine the allele frequency of the target nucleotide;
- and
- f) determining the target polynucleotide is homozygous if the frequency of one allele is 0.7 or greater, or 0.3 or lower, and determining the target polynucleotide is heterozygous if the frequency of one allele is between 0.3 and 0.7.
11 more elements
Dependencies are inferred from the claim text.