Methods and compositions for isolating polynucleotides

摘要

Methods of isolating target double-stranded polynucleotides with internal single-stranded regions are provided. Compositions and kits comprising double-stranded polynucleotides with internal single-stranded regions are also provided.

主权项

1. A method of isolating a double-stranded target polynucleotide molecule, said double-stranded target polynucleotide molecule having an internal destabilized region, wherein the destabilized region comprises (i) one intact polynucleotide strand in the double-stranded target polynucleotide and (ii) another strand substantially complementary to the intact strand and with a plurality of abasic sites or mismatches, said method comprising the steps of:
(a) contacting the internal destabilized region with a single-stranded polynucleotide under appropriate conditions, thereby forming a mixture, said single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion having a sequence sufficiently complementary to the intact polynucleotide strand of the destabilized region of the double-stranded target polynucleotide molecule such that the linker portion hybridizes to the intact polynucleotide strand of the destabilized region of the double-stranded target polynucleotide molecule, and said capture portion has a sequence sufficiently complementary to a nucleotide sequence of a capture substance and does not hybridize to the destabilized region of the double-stranded target polynucleotide molecule, wherein the capture substance comprises a polynucleotide probe having a sequence sufficiently complementary to the capture portion of the single-stranded polynucleotide; (b) adding the capture substance into the mixture so that the capture substance hybridizes with the capture portion of the single-stranded polynucleotide; and (c) isolating the double-stranded target polynucleotide molecule after step (b).