GENOME ANALYSIS USING A NICKING ENDONUCLEASE

• US 20100330556A1
• Filed: 06/30/2009
• Published: 12/30/2010
• Est. Priority Date: 06/30/2009
• Status: Abandoned Application

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Yakhini, Zohar, Hogrefe, Holly, Peter, Brian Jon, Ben-Dor, Amir
• Application Number:
  US12/495,199
• Publication Number:
  US 20100330556A1
• Time in Patent Office: Days
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators

Abstract

A method of genome analysis is provided. In certain embodiments, the method of comprises: a) contacting a genomic sample comprising a double-stranded DNA with a site-specific nicking endonuclease to provide a nicked double-stranded DNA comprising a plurality of nick sites, in which the nicking endonuclease nicks a site adjacent to a variable nucleotide; b) contacting the nicked double-stranded DNA with a polymerase in the presence of a nucleotide composition comprising a first labeled nucleotide comprising a first label, thereby producing a labeled double-stranded DNA that is not labeled at every nick site; c) stretching out the labeled double-stranded DNA to provide a stretched, labeled double-stranded DNA; and d) imaging the stretched, labeled double-stranded DNA to identify a labeling pattern on the stretched labeled double-stranded DNA.

Claims

1. A method of sample analysis comprising:

a) contacting a genomic sample comprising a double-stranded DNA with a site-specific nicking endonuclease to provide a nicked double-stranded DNA comprising a plurality of nick sites, wherein said nicking endonuclease nicks a site adjacent to a variable nucleotide;

b) contacting said nicked double-stranded DNA with a polymerase in the presence of a nucleotide composition comprising a first labeled chain terminator nucleotide comprising a first label, thereby producing a labeled double-stranded DNA in which not every nick site is labeled with said first label;

c) stretching out said labeled double-stranded DNA to provide a stretched, labeled double-stranded DNA; and

d) imaging said stretched, labeled double-stranded DNA to identify a labeling pattern on said stretched labeled double-stranded DNA.

2. The method of claim 1, wherein said nucleotide composition comprises said first labeled chain terminator nucleotide and no other labeled nucleotides.

3. The method of claim 1, further comprising recording said labeling pattern as a sequence of distances between labeled nick sites along said stretched, labeled double-stranded DNA.

4. The method of claim 1, wherein said nucleotide composition comprises at least:

a) said first labeled chain terminator nucleotide comprising a first label; and

b) a second labeled chain terminator nucleotide comprising a second label, wherein said first label and said second label emit distinguishable colors.

5. The method of claim 4, further comprising recording said labeling pattern as a sequence of colors of labeled nick sites along said stretched, double-stranded DNA, wherein said colors are emitted by labeled chain terminator nucleotides at said nick sites.

6. The method of claim 4, further comprising recoding said labeling pattern as a sequence of colors of labeled nick sites and distances between said labeled nick sites along said stretched, labeled double-stranded DNA, wherein said colors are emitted by labeled chain terminator nucleotides at said nick sites.

7. The method of claim 4, wherein said nucleotide composition comprises:

a) said first labeled chain terminator nucleotide comprising a first label;

b) said second labeled chain terminator nucleotide comprising a second label;

c) a third labeled chain terminator nucleotide comprising a third label; and

d) a fourth labeled chain terminator nucleotide comprising a fourth label, wherein said first label, said second label, said third label, and said fourth label emit different colors.

8. The method of claim 1, wherein said labeling pattern identifies said double-stranded DNA as being a specific genomic region.

9. The method of claim 1, wherein said method further comprises

e) comparing said labeling pattern to a reference pattern.

10. The method of claim 1, wherein said polymerase incorporates said labeled chain terminator nucleotide in a position 5′

to said nick site.

11. The method of claim 1, wherein said polymerase incorporate said labeled chain terminator in a position 3′

to said nick site.

12. The method of claim 1, wherein said polymerase in contacting step comprises a 3′

to 5′

exonuclease activity in addition to a polymerase activity.

13. The method of claim 1, wherein said genomic sample comprises double-stranded DNA is contacted with a ligase prior to said contacting step a).

14. The method of claim 1, further comprising labeling a backbone of said double-stranded DNA to produced a labeled DNA backbone prior to imaging step d).

15. The method of claim 1, wherein said polymerase comprises a mixture of a plurality of enzymes.

16. The method of claim 1, wherein said labeled chain terminator nucleotide is a phosphorothioated nucleotide analog.

17. The method of claim 1, wherein said labeled chain terminator nucleotide is an acyclo-nitrogenous base.

18. The method of claim 1, wherein said double-stranded DNA is at least about 50 kilobases long.

19. The method of claim 12, wherein said polymerase is engineered to have an enhanced exonuclease activity, relative to the polymerase activity.

20. A system for sample analysis comprising:

a) reagents for performing the method of claim 1, wherein said reagents comprise a site specific nicking endonuclease that nicks sites adjacent to variable nucleotide, and a nucleotide composition comprising a labeled chain terminator nucleotide;

b) a stretching device;

c) an imaging workstation;

d) a computer for recording;

e) a computer-readable medium comprising a database of reference patterns.