Method of haplotyping and kit therefor
First Claim
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1. A method of identifying the haplotype of an organism, the method comprising:
- (a) providing a sample comprising nucleic acids from the organism, wherein the nucleic acids comprise at least two copies of an isogenic nucleotide sequence of interest, (b) aliquotting the nucleic acids into test locations such that at least one test location is expected to contain one, and only one, isogenic nucleotide sequence of interest, (c) amplifying the isogenic nucleotide sequence of interest in a predetermined number of test locations to create amplification products, (i) wherein amplifying the isogenic nucleotide sequence of interest employs two pairs of oligonucleotide primers (ii) such that at least one test location is expected to contain amplification products having a unique nucleotide sequence corresponding to the nucleotide sequence of one, and only one, of the isogenic nucleotide sequences of interest in the organism'"'"'s genome, and (d) detecting the presence or absence of specific forms of a first nucleotide polymorphism and a second nucleotide polymorphism in the isogenic region of interest at two non-contiguous positions in the nucleotide sequence of interest by detecting the presence or absence of specific forms of the first nucleotide polymorphism and second nucleotide polymorphism in the amplification products in each of the predetermined number of test locations comprising amplified nucleic acids, such that the haplotype of the organism is identified.
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Abstract
A method of identifying the haplotype of an organism comprising the use of multiple duplexed amplifications of a single copy of an isogenic nucleotide sequence of interest coupled with detection of putative nucleotide sequence polymorphisms, as well as a kit and apparatus therefor.
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Citations
22 Claims
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1. A method of identifying the haplotype of an organism, the method comprising:
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(a) providing a sample comprising nucleic acids from the organism, wherein the nucleic acids comprise at least two copies of an isogenic nucleotide sequence of interest, (b) aliquotting the nucleic acids into test locations such that at least one test location is expected to contain one, and only one, isogenic nucleotide sequence of interest, (c) amplifying the isogenic nucleotide sequence of interest in a predetermined number of test locations to create amplification products, (i) wherein amplifying the isogenic nucleotide sequence of interest employs two pairs of oligonucleotide primers (ii) such that at least one test location is expected to contain amplification products having a unique nucleotide sequence corresponding to the nucleotide sequence of one, and only one, of the isogenic nucleotide sequences of interest in the organism'"'"'s genome, and (d) detecting the presence or absence of specific forms of a first nucleotide polymorphism and a second nucleotide polymorphism in the isogenic region of interest at two non-contiguous positions in the nucleotide sequence of interest by detecting the presence or absence of specific forms of the first nucleotide polymorphism and second nucleotide polymorphism in the amplification products in each of the predetermined number of test locations comprising amplified nucleic acids, such that the haplotype of the organism is identified. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)
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19. A kit, useful for identifying the haplotype of an organism having a diploid genome, comprising:
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(a) a first pair of oligonucleotides, (b) a second pair of oligonucleotides, wherein (i) the first pair of oligonucleotides are complementary to a nucleotide sequence flanking a first polymorphism in an isogenic region of the organism'"'"'s genome (ii) the second pair of oligonucleotides are complementary to a nucleotide sequence flanking a second polymorphic site in an isogenic region of the organism'"'"'s genome (iii) no oligonucleotide of the first pair or second pair of oligonucleotides is complementary to a nucleotide sequence in the isogenic nucleotide sequence of interest that is complementary to another oligonucleotide of the first pair or second pair of oligonucleotides (c) a first probe specific for the first polymorphism within a first isogenic nucleotide sequence of interest, and (d) a second probe specific for the second polymorphism within a second isogenic nucleotide sequence of interest. - View Dependent Claims (20, 21)
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22. A kit, useful for identifying the haplotype of an organism having a diploid genome, comprising:
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(a) a first pair of oligonucleotides, (b) a second pair of oligonucleotide, wherein (i) the first pair of oligonucleotides are complementary to a nucleotide sequence flanking a first polymorphism in an isogenic region of the organism'"'"'s genome (ii) the second pair of oligonucleotides are complementary to a nucleotide sequence flanking a second polymorphic site in an isogenic region of the organism'"'"'s genome (iii) no oligonucleotide of the first pair or second pair of oligonucleotides is complementary to a nucleotide sequence in the isogenic nucleotide sequence of interest that is complementary to another oligonucleotide of the first pair or second pair of oligonucleotides (c) a means of detecting one or more specific forms of a first polymorphism within a first isogenic nucleotide sequence of interest, and (d) a means of detecting one or more specific forms of a second polymorphism within a second isogenic nucleotide sequence of interest.
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Specification