Genomic library construction

• US 7,985,546 B2
• Filed: 11/05/2008
• Issued: 07/26/2011
• Est. Priority Date: 05/18/2006
• Status: Expired due to Fees

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Zhang, Kun, Church, George M.
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US12/265,184
• Publication Number:
  US 20090137407A1
• Time in Patent Office: 993 Days
• Field of Search: 435/6, 435 91- 2, 536/24.33
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease

Abstract

Compositions and methods for amplifying nucleic acid sequences from a single cell are provided. Compositions and methods for constructing a genomic library from a single cell are also provided.

Claims

1. A method of amplifying a nucleic acid sequence from a single cell comprising:

amplifying a nucleic acid sequence from a single cell to produce an amplified nucleic acid sequence; and

contacting said amplified nucleic acid sequence with phi-29 DNA polymerase, S1 nuclease and a DNA polymerase having a 5′

exonuclease activity.

2. The method of claim 1, wherein said nucleic acid sequence is genomic DNA.

3. The method of claim 1, wherein said single cell is a bacterial cell.

4. The method of claim 1, wherein said amplifying is by multiple displacement amplification.

5. The method of claim 1, wherein said contacting occurs in the following order:

a) contacting said amplified nucleic acid sequence with phi-29 DNA polymerase;

b) contacting said amplified nucleic acid sequence with S1 nuclease; and

c) contacting said amplified nucleic acid sequence with a DNA polymerase having a 5′

exonuclease activity.

21. The method of claim 1 wherein the DNA polymerase is DNA polymerase I.

6. A method of reducing hyperbranched nucleic acid sequences in an amplified nucleic acid sequence from a single cell comprising:

amplifying a nucleic acid sequence from the single cell to produce an amplified nucleic acid sequence; and

contacting said amplified nucleic acid sequence with phi-29 DNA polymerase, S1 nuclease and a DNA polymerase having a 5′

exonuclease activity to reduce hyperbranched nucleic acid sequences in the amplified nucleic acid sequence,

such that the amount of hyperbranched nucleic acid sequences in the amplified nucleic acid sequence after contacting is reduced relative to the amount of hyperbranched nucleic acid sequences in the amplified nucleic acid sequence before contacting.

7. The method of claim 6, wherein said nucleic acid sequence is genomic DNA.

8. The method of claim 6, wherein said amplifying is by multiple displacement amplification.

9. The method of claim 6, wherein said single cell is a prokaryotic cell or a eukaryotic cell.

10. The method of claim 9, wherein said prokaryotic cell is a bacterial cell.

11. The method of claim 9, wherein said eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell and a mammalian cell.

22. The method of claim 6 wherein the DNA polymerase is DNA polymerase I.

12. A method of reducing an amount of chimeric nucleic acid sequences present in cloned DNA comprising:

amplifying a nucleic acid sequence to produce an amplified nucleic acid sequence;

contacting said amplified nucleic acid sequence with phi-29 DNA polymerase, S1 nuclease and a DNA polymerase having a 5′

exonuclease activity;

placing said amplified nucleic acid sequence into a vector; and

cloning said vector,

such that the amount of chimeric nucleic acid sequences present in the amplified nucleic acid sequence after contacting is reduced relative to the amount of chimeric nucleic acid sequences present in the amplified nucleic acid sequence before contacting.

13. The method of claim 12, wherein said nucleic acid sequence that is amplified is genomic DNA.

14. The method of claim 12, wherein said amplifying is by multiple displacement amplification.

23. The method of claim 12 wherein the DNA polymerase is DNA polymerase I.

15. A method of constructing a genomic library comprising:

obtaining genomic DNA from a single cell;

amplifying said genomic DNA to produce amplified genomic DNA;

contacting said amplified genomic DNA with phi-29 DNA polymerase, S1 nuclease and a DNA polymerase having a 5′

exonuclease activity;

placing said amplified genomic DNA into two or more vectors; and

transforming each of said vectors into a competent cell.

16. The method of claim 15, wherein said amplifying is by multiple displacement amplification.

17. The method of claim 15, wherein said single cell is a prokaryotic cell or a eukaryotic cell.

18. The method of claim 17, wherein said prokaryotic cell is a bacterial cell.

19. The method of claim 17, wherein said eukaryotic cell is selected from the group consisting of a yeast cell, an insect cell and a mammalian cell.

20. A method of determining genome diversity between two cells comprising:

constructing a first genomic library according to claim 15 for a first cell;

constructing a second genomic library according to claim 15 for a second cell;

sequencing at least a portion of the first genomic library to obtain a first genomic sequence;

sequencing at least a portion of the second genomic library to obtain a second genomic sequence; and

comparing the first genomic sequence and the second genomic sequence to determine genome diversity.

24. The method of claim 15 wherein the DNA polymerase is DNA polymerase I.