METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACID SEQUENCING LIBRARIES
Methods and compositions are provided for preparing DNA libraries. Enzymes, adaptors, and sample nucleic acids are provided in a single reaction mixture to facilitate library preparation.
1 .- 36 . (canceled)
37 . A kit for preparing a nucleic acid library, comprising:
(i) a nucleic acid adaptor, comprising a first nucleic acid strand comprising a first 5′ end and a first 3′ end and a second nucleic acid strand comprising a second 5′ end and a second 3′ end,
wherein each adaptor comprises a double stranded region, a first single stranded region comprising the first 5′ end, and a second single stranded region comprising the second 3′ end,
wherein the second 3′ end comprises a modification to prevent extension with a DNA polymerase enzyme;
(ii) a proofreading DNA polymerase enzyme, wherein the proofreading DNA polymerase enzyme degrades 3′ overhang sequences, if any, and fills in 5′ overhang sequences, if any, on DNA duplex fragments that comprise first and second strands, thereby producing blunt ended DNA duplex fragments comprising first and second ends, wherein each of the first and second strands of the DNA duplex comprises a 5′ terminal nucleotide, and wherein the proofreading DNA polymerase enzyme degrades 3′ overhang sequences, if any, and fills in 5′ overhang sequences, if any, on the adaptors, producing adaptors with blunt ended double stranded regions comprising 5′ terminal nucleotides;
(iii) a polynucleotide kinase enzyme that phosphorylates the 5′ terminal nucleotides on the first and second strands of said DNA duplex fragments and/or on the double stranded regions of the adaptors; and
(iv) a ligase enzyme that joins said adaptor double stranded regions to each of the first and second ends of the DNA duplex fragments, thereby producing adaptor ligated DNA duplex fragments.
38 .- 39 . (canceled)
40 . The kit of claim 37 , wherein the nucleic acid adaptor comprises a 5′ overhang that comprises the second 5′ end.
41 . The kit of claim 37 , wherein the double stranded region of the nucleic acid adaptor is blunt ended and comprises a 5′ terminal phosphate.
42 . The kit of claim 37 , wherein the double stranded region of the nucleic acid adaptor is blunt ended and comprises an unphosphorylated 5′ terminal nucleotide.
43 . The kit of claim 37 , wherein the nucleic acid adaptor comprises a modification at or near the first 3′ end to prevent degradation by an exonuclease enzyme.
44 . The kit of claim 37 , wherein the first 5′ end of the nucleic acid adaptor comprises a modification to prevent phosphorylation and ligation.
45 . The kit of claim 37 , wherein the first 3′ end of the nucleic acid adaptor comprises a modification to prevent degradation by an exonuclease enzyme, and wherein the first 5′ end of the nucleic acid adaptor comprises a modification to prevent phosphorylation and ligation.
46 . A reaction mixture comprising:
(i) a plurality of DNA duplex fragments comprising first and second nucleic acid strands;
(ii) a nucleic acid adaptor, comprising a first nucleic acid strand comprising a first 5′ end and a first 3′ end and a second nucleic acid strand comprising a second 5′ end and a second 3′ end,
wherein each adaptor comprises a double stranded region, a first single stranded region comprising the first 5′ end, and a second single stranded region comprising the second 3′ end,
wherein the second 3′ end comprises a modification to prevent extension with a DNA polymerase enzyme;
(iii) a proofreading DNA polymerase enzyme,
wherein the proofreading DNA polymerase enzyme degrades 3′ overhang sequences, if any, and fills in 5′ overhang sequences, if any, on the DNA duplex fragments, thereby producing blunt ended DNA duplex fragments comprising first and second ends, wherein each of the first and second strands of the DNA duplex comprises a 5′ terminal nucleotide, and wherein the proofreading DNA polymerase enzyme degrades 3′ overhang sequences, if any, and fills in 5′ overhang sequences, if any, on the adaptors, producing adaptors with blunt ended double stranded regions comprising 5′ terminal nucleotides;
(iv) a polynucleotide kinase enzyme that phosphorylates the 5′ terminal nucleotides on the first and second strands of said DNA duplex fragments and/or on the double stranded regions of the adaptors; and
(v) a ligase enzyme that joins said adaptor double stranded regions to each of the first and second ends of the DNA duplex fragments, thereby producing adaptor ligated DNA duplex fragments.
47 . A reaction mixture of claim 46 , comprising a temperature that is appropriate for activity of the proofreading DNA polymerase enzyme, the polynucleotide kinase enzyme, and the ligase enzyme.
48 . The reaction mixture of claim 46 , wherein the nucleic acid adaptor comprises a 5′ overhang that comprises the second 5′ end.
49 . The reaction mixture of claim 46 , wherein the double stranded region of the nucleic acid adaptor is blunt ended and comprises a 5′ terminal phosphate.
50 . The reaction mixture of claim 46 , wherein the double stranded region of the nucleic acid adaptor is blunt ended and comprises an unphosphorylated 5′ terminal nucleotide.
51 . The reaction mixture of claim 46 , wherein the nucleic acid adaptor comprises a modification at or near the first 3′ end to prevent degradation by an exonuclease enzyme.
52 . The reaction mixture of claim 46 , wherein the first 5′ end of the nucleic acid adaptor comprises a modification to prevent phosphorylation and ligation.
53 . The reaction mixture of claim 46 , wherein the first 3′ end of the nucleic acid adaptor comprises a modification to prevent degradation by an exonuclease enzyme, and wherein the first 5′ end of the nucleic acid adaptor comprises a modification to prevent phosphorylation and ligation.