Methods and Compositions for Isolating Polynucleotides

Abstract

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.

What is claimed is: 
     
         1 . A method of selecting a target double-stranded polynucleotide molecule, said target have an internal destabilized region, wherein the destabilized region comprises (i) an intact polynucleotide strand in the target double-stranded polynucleotide and (ii) a 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, said single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion having a sequence sufficiently complementary to hybridize to the intact polynucleotide strand of the destabilized region of the target under appropriate conditions, and said capture portion does not hybridize to the destabilized region of the target; and   (b) contacting the target with a capture substance that interacts with the capture portion of the single-stranded polynucleotide to select the target.   
     
     
         2 . A method of selecting a target double-stranded polynucleotide molecule, said target having an internal single-stranded region, said method comprising the steps of:
 (a) contacting the internal single-stranded region with a single-stranded polynucleotide, said single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion having a sequence sufficiently complementary to hybridize to the single-stranded region of the target under appropriate conditions, and said capture portion does not hybridize to the single-stranded region of the target; and   (b) contacting the target with a capture substance that interacts with the capture portion of the single-stranded polynucleotide to select the target,   wherein the single stranded polynucleotide is not ligated to the target.   
     
     
         3 . A method of selecting a target double-stranded polynucleotide molecule, said target having an internal single-stranded region, said method comprising the steps of:
 (a) generating the single-stranded region in the target double-stranded polynucleotide molecule by a process selected from the group consisting of nick-translation DNA synthesis and nick-mediated strand-displacement DNA synthesis,   (b) contacting the internal single-stranded region with a single-stranded polynucleotide, said single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion having a sequence sufficiently complementary to hybridize to the single-stranded region of the target under appropriate conditions, and said capture portion does not hybridize to the single-stranded region of the target; and   (c) contacting the target with a capture substance that interacts with the capture portion of the single-stranded polynucleotide to select the target.   
     
     
         4 . The method of  claim 1 ,  2 , or  3  wherein the single-stranded polynucleotide is allele-specific. 
     
     
         5 . The method of  claim 1 ,  2 , or  3  wherein the single-stranded polynucleotide is haplotype-specific. 
     
     
         6 . The method of any one of  claims 2  to  5  wherein the internal single-stranded region is a flap. 
     
     
         7 . The method of any one of  claims 2  to  5  wherein the internal single-stranded region is a gap. 
     
     
         8 . The method of any of  claims 1  to  7  further comprising the step of isolating the target polynucleotide molecule selected in step (b). 
     
     
         9 . The method of any of  claims 1  to  8  further comprising the step of extending the capture portion and/or the linker portion of the single-stranded polynucleotide. 
     
     
         10 . The method of  claim 9  wherein extending the single-stranded polynucleotide adds a specific sequence. 
     
     
         11 . The method of  claim 10  wherein the single-stranded polynucleotide is extended by rolling circle amplification. 
     
     
         12 . The method of any of  claims 9  to  11  wherein extending the single-stranded polynucleotide adds a sequence that interacts with the capture substance. 
     
     
         13 . The method of any one of  claims 1  to  12  further comprising the step of cleaving a phosphodiester bond of the target polynucleotide molecule. 
     
     
         14 . The method of any one of  claim 1 ,  2  or  4 - 10 , further comprising the step of generating the single-stranded region in the target double-stranded polynucleotide molecule by cleaving a phosphodiester bond of the target double-stranded polynucleotide molecule and removing one or more bases in a single strand of the target polynucleotide molecule adjacent the phosphodiester bond that was cleaved. 
     
     
         15 . The method of any one of  claim 1 ,  4 ,  5 , or  8 - 14 , further comprising the step of generating the internal destabilized region by nick-translation DNA synthesis and treating with Uracil-DNA glycosylase (UDG). 
     
     
         16 . The method of any one of  claim 2  or  6 - 14 , further comprising the step of generating the single-stranded region in the target double-stranded polynucleotide molecule by a process selected from the group consisting of nick-mediated exonuclease DNA degradation, nick-translation DNA synthesis, and nick-mediated strand-displacement DNA synthesis, after the phosphodiester bond is cleaved. 
     
     
         17 . The method of any one of  claims 1  to  16  wherein the target polynucleotide molecule is released from the capture substance. 
     
     
         18 . The method of  claim 17  wherein the target double-stranded polynucleotide molecule is released by enzymatic degradation of the capture portion and capture substance. 
     
     
         19 . The method of any one of  claims 13  to  18  wherein the phosphodiester bond is cleaved by a nicking endonuclease. 
     
     
         20 . The method of  claim 19  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. 
     
     
         21 . The method of  claim 19  or  20  wherein the nicking endonuclease is heat inactivated or removed after the phosphodiester bond is cleaved. 
     
     
         22 . The method of any of  claims 1  through  21  wherein the capture substance comprises a polynucleotide probe having a sequence sufficiently complementary to hybridize to the capture portion of the single-stranded polynucleotide. 
     
     
         23 . The method of  claim 22  wherein the polynucleotide probe hybridizes to the sequence added to the single stranded polynucleotide. 
     
     
         24 . The method of  claim 22  or  23  wherein the polynucleotide probe is covalently attached to a bead. 
     
     
         25 . The method of any one of  claims 1  to  24  wherein the capture substance comprises:
 (a) a polynucleotide probe having a sequence sufficiently complementary to hybridize to the capture portion of the single-stranded polynucleotide, said probe covalently attached to a first binding partner; and 
 (b) a second binding partner. 
 
     
     
         26 . The method of  claim 25  wherein the second binding partner is attached to a bead. 
     
     
         27 . The method of  claim 25  or  26  wherein the first binding partner comprises biotin and the second binding partner comprises streptavidin. 
     
     
         28 . The method of any one of  claims 24  to  27  wherein the bead is magnetic. 
     
     
         29 . The method of any one of  claims 1  to  28  wherein the capture substance is immobilized on a solid support. 
     
     
         30 . The method of  claim 29  wherein the solid support is selected from the group consisting of a polypropylene tube, a capillary tube, and a glass slide. 
     
     
         31 . A composition comprising a substantially double-stranded polynucleotide molecule with an internal destabilized region that is associated with a single-stranded polynucleotide, said destabilized region comprising (i) an intact polynucleotide strand in the target double-stranded polynucleotide and (ii) a strand substantially complementary to the intact strand and with a plurality of abasic sites or mismatches, said single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion sufficiently complementary to the intact strand of the destabilized region to allow the linker portion and the intact strand to hybridize, and said capture portion not complementary to the substantially double-stranded polynucleotide molecule. 
     
     
         32 . The composition of  claim 31  further comprising a polynucleotide probe hybridized to the capture portion of the single-stranded polynucleotide. 
     
     
         33 . The composition of  claim 32  further comprising a bead. 
     
     
         34 . The composition of  claim 33  wherein the polynucleotide probe is covalently attached to the bead. 
     
     
         35 . The composition of  claim 34  wherein the bead is magnetic. 
     
     
         36 . The composition of  claim 32  wherein the polynucleotide probe is biotinylated. 
     
     
         37 . The composition of  claim 36  further comprising a streptavidin-coated bead. 
     
     
         38 . A DNA selection kit comprising: (a) a single-stranded polynucleotide comprising a linker portion and a capture portion, said linker portion having a sequence sufficiently complementary to hybridize to a single-stranded region of a substantially double-stranded target polynucleotide molecule under appropriate conditions, and said capture portion not complementary to the single-stranded region of the target; (b) a capture substance that interacts with the capture portion of the single-stranded polynucleotide; and (c) a DNA polymerase. 
     
     
         39 . The kit of  claim 38  further comprising a nicking endonuclease. 
     
     
         40 . The kit of  claim 39  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. 
     
     
         41 . The kit of any one of  claims 38  to  40  further comprising an exonuclease. 
     
     
         42 . The kit of  claim 41  wherein the exonuclease is a 5′ exonuclease or a 3′ exonuclease. 
     
     
         43 . The kit of any one of  claims 38  to  42  further comprising a DNA polymerase. 
     
     
         44 . The kit of any of  claims 38  to  43  further comprising a dNTP mix, said dNTP mix comprising dTTP, dATP, dCTP, and dGTP. 
     
     
         45 . The kit of any of  claims 38  to  43  further comprising a dNTP mix, said dNTP mix comprising dUTP, dATP, dCTP, and dGTP. 
     
     
         46 . The kit of  claim 45  further comprising dTTP. 
     
     
         47 . The kit of any of  claims 38  to  46  further comprising a uracil-DNA Glycosylase (UDG) enzyme and an abasic endonuclease. 
     
     
         48 . The kit of  claim 47  wherein the abasic endonuclease is selected from the group consisting of APE1, endonuclease III(Nth), endonuclease IV, endonuclease VIII, T4 endonuclease V, Tma endonuclease III, and Tth endonuclease IV. 
     
     
         49 . The kit of any one of  claims 38  to  48  wherein the capture substance comprises a polynucleotide probe having a sequence sufficiently complementary to hybridize to the capture portion of the single-stranded polynucleotide. 
     
     
         50 . The kit of  claim 49  wherein the polynucleotide probe is covalently attached to a bead. 
     
     
         51 . The kit of  claim 49  wherein the polynucleotide probe is covalently attached to biotin. 
     
     
         52 . The kit of  claim 51  wherein the capture substance further comprises a streptavidin-coated bead. 
     
     
         53 . The kit of  claim 50  or  52  wherein the bead is magnetic. 
     
     
         54 . A method of selecting a target double-stranded polynucleotide molecule, said target having an internal single-stranded region, said method comprising the step of:
 (a) generating the single-stranded region in the target double-stranded polynucleotide molecule by nick-mediated strand-displacement DNA synthesis   (b) contacting the internal single-stranded region with a capture substance that interacts with the internal single-stranded polynucleotide to select the target.   
     
     
         55 . The method of  claim 54  wherein the internal single-stranded region is a flap. 
     
     
         56 . The method of  claim 54  or  55  further comprising the step of isolating the target polynucleotide molecule. 
     
     
         57 . The method of any of  claims 54  to  56  further comprising the step of extending the single-stranded region. 
     
     
         58 . The method of  claim 57  wherein extending the single-stranded region adds a specific sequence. 
     
     
         59 . The method of  claim 57  or  58  wherein extending the single-stranded region adds a sequence that interacts with the capture substance. 
     
     
         60 . The method of any of  claims 57  to  59  wherein the single-stranded region is extended by rolling circle amplification. 
     
     
         61 . The method of any one of  claims 54  to  60  further comprising the step of cleaving a phosphodiester bond of the target polynucleotide molecule. 
     
     
         62 . The method of any one of  claims 54  to  61  wherein the target polynucleotide molecule is released from the capture substance. 
     
     
         63 . The method of  claim 62  wherein the target double-stranded polynucleotide molecule is released by enzymatic degradation of the capture portion and capture substance. 
     
     
         64 . The method of  claim 61  wherein the phosphodiester bond is cleaved by a nicking endonuclease. 
     
     
         65 . The method of  claim 64  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. 
     
     
         66 . The method of  claim 64  or  65  wherein the nicking endonuclease is heat inactivated or removed after the phosphodiester bond is cleaved. 
     
     
         67 . The method of any of  claims 54  through  66  wherein the capture substance comprises a polynucleotide probe having a sequence sufficiently complementary to hybridize to the internal single-stranded region. 
     
     
         68 . The method of any of  claims 57  to  67  wherein the polynucleotide probe hybridizes to the sequence added to the single stranded polynucleotide. 
     
     
         69 . The method of  claim 67  or  68  wherein the polynucleotide probe is covalently attached to a bead. 
     
     
         70 . The method of any one of  claims 54  to  69  wherein the capture substance comprises:
 (a) a polynucleotide probe having a sequence sufficiently complementary to hybridize to the internal single-stranded region, said probe covalently attached to a first binding partner; and 
 (b) a second binding partner. 
 
     
     
         71 . The method of  claim 70  wherein the second binding partner is attached to a bead. 
     
     
         72 . The method of  claim 70  or  71  wherein the first binding partner comprises biotin and the second binding partner comprises streptavidin. 
     
     
         73 . The method of any one of  claims 69  to  72  wherein the bead is magnetic. 
     
     
         74 . The method of any one of  claims 62  to  73  wherein the capture substance is immobilized on a solid support. 
     
     
         75 . The method of  claim 74  wherein the solid support is selected from the group consisting of a polypropylene tube, a capillary tube, and a glass slide.