Methods and compositions for isolating polynucleotides
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.
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).
2. The method of claim 1 , wherein the single-stranded polynucleotide is allele-specific.
3. The method of claim 1 , wherein the single-stranded polynucleotide is haplotype-specific.
4. The method of claim 1 , further comprising the step of extending the capture portion of the single-stranded polynucleotide after step (a).
5. The method of claim 4 , wherein the extending step adds a specific sequence to the capture portion of the single-stranded polynucleotide.
6. The method of claim 5 , wherein the capture portion of the single-stranded polynucleotide is extended by rolling circle amplification.
7. The method of claim 5 , wherein the polynucleotide probe hybridizes to the specific sequence.
8. The method of claims 4 , wherein the extending step adds a sequence to the capture portion.
9. The method of claim 1 , further comprising a step of cleaving a phosphodiester bond of the double-stranded target polynucleotide molecule after step c).
10. The method of claim 9 , wherein the phosphodiester bond is cleaved by a nicking endonuclease.
11. The method of claim 10 wherein the nicking endonuclease is selected from the group consisting of Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI and Nt.CviPII.
12. The method of claim 1 , further comprising a step of generating the single-stranded region in the double-stranded target polynucleotide molecule by cleaving a phosphodiester bond of the double-stranded target polynucleotide molecule and removing one or more bases in a single strand of the double-stranded target polynucleotide molecule adjacent the phosphodiester bond after step c).
13. The method of claim 1 , further comprising a step of generating the double-stranded target polynucleotide molecule before step a) by cleaving a phosphodiester bond in a double-stranded polynucleotide molecule with a nicking endonuclease, producing a nicked double-stranded target polynucleotide molecule, incubating the nicked double-stranded polynucleotide molecule with dGTP, dATP, dCTP, and dUTP nucleotides and a DNA polymerase with 5′ to 3′ exonuclease activity thereby synthesizing a new double-stranded polynucleotide molecule, and treating the synthesized double-stranded polynucleotide molecule with Uracil-DNA glycosylase (UDG).
14. The method of claim 1 , wherein the double-stranded target polynucleotide molecule is released from the capture substance during step (c).
15. The method of claim 14 wherein the target double-stranded polynucleotide molecule is released by enzymatic degradation of the capture portion and capture substance.
16. The method of claim 1 , wherein the polynucleotide probe is covalently attached to a bead.
17. The method of claim 1 , wherein the polynucleotide probe is covalently attached to a first binding partner and a second binding partner.