Allele-specific sequence variation analysis
First Claim
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1. A method for haplotyping a plurality of polymorphic markers, comprising:
- a) amplifying a target DNA molecule, using two primers, wherein one of the primers is allele-specific, and wherein at least one of the primers comprises an RNA promoter in the presence ofi. a competitor oligonucleotide comprising a modified 3′
-end nucleotide selected from an inverted base, a dideoxynucleotide bound to the penultimate nucleotide by a methylphosphonate link, a ribonucleotide, a 3′
Phosphate, a carbon chain spacer, or a peptide nucleic acid (PNA), wherein the competitor is an extension inhibitor of an unwanted allele, andii. a mixture of thermostable DNA polymerases, wherein at least one of the polymerases comprises 3′
-5′
exonuclease activity, andobtaining a double stranded DNA molecule;
b) fragmenting the double stranded DNA molecule, or an RNA product transcribed from the double stranded DNA molecule, at a plurality of specific and predictable sites;
c) fragmenting or simulating fragmentation of a reference nucleic acid into fragments at the same plurality of specific and predictable sites of step b);
d) comparing the fragments of step b) with the fragments or simulated fragments of step c); and
e) from the comparison of fragments of d), haplotyping a plurality of linked SNPs on the target DNA molecule corresponding to a single or specific allele at one or more loci, in a single reaction.
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Abstract
Fragmentation-based methods and systems, particularly mass spectrometry based methods and systems, for the analysis of sequence variations including haplotypes are provided. Also provided are methods for obtaining a specific allele from a nucleic acid mixture, such as genomic DNA.
175 Citations
26 Claims
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1. A method for haplotyping a plurality of polymorphic markers, comprising:
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a) amplifying a target DNA molecule, using two primers, wherein one of the primers is allele-specific, and wherein at least one of the primers comprises an RNA promoter in the presence of i. a competitor oligonucleotide comprising a modified 3′
-end nucleotide selected from an inverted base, a dideoxynucleotide bound to the penultimate nucleotide by a methylphosphonate link, a ribonucleotide, a 3′
Phosphate, a carbon chain spacer, or a peptide nucleic acid (PNA), wherein the competitor is an extension inhibitor of an unwanted allele, andii. a mixture of thermostable DNA polymerases, wherein at least one of the polymerases comprises 3′
-5′
exonuclease activity, andobtaining a double stranded DNA molecule; b) fragmenting the double stranded DNA molecule, or an RNA product transcribed from the double stranded DNA molecule, at a plurality of specific and predictable sites; c) fragmenting or simulating fragmentation of a reference nucleic acid into fragments at the same plurality of specific and predictable sites of step b); d) comparing the fragments of step b) with the fragments or simulated fragments of step c); and e) from the comparison of fragments of d), haplotyping a plurality of linked SNPs on the target DNA molecule corresponding to a single or specific allele at one or more loci, in a single reaction. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)
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Specification