| 摘要 |
Provided is an in vitro method, termed ChIp-DASL, to examine the binding of proteins to DNA, in particular to regulatory regions, across a genome or a portion thereof, e.g. a chromosome. The method steps comprise: a) immunoprecipitating a polynucleotide associated with a polypeptide to obtain an enriched polynucleotide preparation; b) disassociating the polynucleotide and polypeptide in the enriched preparation; c) contacting the polynucleotide with a primer pair under conditions whereby the primer pair hybridizes to the polynucleotide to form a first hybridization complex, each primer comprising at least two portions, a first portion comprising a target specific oligonucleotide that is capable of hybridizing to a target polynucleotide in the enriched preparation, and a second portion comprising a universal primer landing site, the two primers being specific for an upstream and downstream segment of the target polynucleotide, wherein the universal landing sites are not the same; d) contacting the first hybridization complex with a ligase under conditions whereby primer pairs hybridized to the polynucleotide are ligated to form a ligated probe; e) amplifying the ligated probe with universal primers to generate an amplified-labeled product; f) contacting the amplified-labeled product with an array of oligonucleotides under conditions whereby the ligated probe hybridizes to a complementary oligonucleotide in the array to form assay complexes; and g) detecting the assay complexes, wherein the presence of complexes is indicative of DNA that binds the immunoprecipitated polypeptide.
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