| 主权项 |
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 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, and obtaining 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. |
