Methods for Genotyping

摘要

Novel methods and kits are disclosed for reducing the complexity of a nucleic acid sample to interrogate a collection of target sequences, for example, to discriminating between alleles at polymorphic positions in a genome. Complexity reduction can be accomplished by extension of a capture probes followed by amplification of the extended capture probe using common primers. The capture probes may be locus specific and allele-specific. The amplified sample may be hybridized to an array designed to interrogate the desired fragments for the presence or absence of a polymorphism. In some aspects the methods employ allele-specific extension of oligonucleotides that are complementary to one of the alleles at the 3′ end of the oligonucleotide. The allele-specific oligonucleotides are resistant to proof reading activity from a polymerase and may be extended in an allele-specific manner by a DNA polymerase with a functional 3′ to 5′ exonuclease activity.

主权项

1. A method of amplifying a collection of target sequences from a nucleic acid sample the method comprising:
fragmenting the nucleic acid sample; ligating an adaptor comprising a second common sequence to the fragments, wherein the adaptor is ligated to the fragments so that the strand that is ligated to the 5′ end of the fragment strands comprises the second common sequence and the strand that is ligated to the 3′ end of the fragments lacks the complement of the second common sequence and is blocked from extension at the 3′ end; hybridizing the adaptor-ligated fragments to a collection of capture probes comprising a plurality of different species of primers wherein each species comprises a first common sequence that is common to each capture probe in the collection of capture probes and a 3′ variable region that is specific for a target sequence in the collection of target sequences; extending the capture probes to obtain extension products; and amplifying the extended capture probes with first and second common sequence primers to obtain an amplified collection of target sequences.