Method for identification of genetic markers

• US 7,300,751 B2
• Filed: 09/06/2002
• Issued: 11/27/2007
• Est. Priority Date: 06/30/2000
• Status: Expired due to Fees

• Current Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Original Assignee: Syngenta Participations AG (China National Chemical Corporation)
• Inventors: Wang, Xun, Shi, Liang, Li, Bi-Yu
• Primary Examiner(s): Chunduru; Suryaprabha
• Application Number:
  US10/236,363
• Publication Number:
  US 20030165923A1
• Time in Patent Office: 1,908 Days
• Field of Search: 435/6, 435/91.2, 536/22.1, 536/24.33, 536/23.1
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

Methods are provided for identifying genetic markers and for determining methylation patterns using restriction endonucleases having degenerate recognition or cleavage sequences.

Claims

1. A method for identifying genetic markers comprising:

(a) obtaining genomic DNA from a first source having a first phenotype or trait of interest;

(b) digesting said DNA with at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base, wherein;

(i) said degenerate sequence is represented by the formula N^m, where N is the extent of degeneracy, and m is the number of degenerate bases; and

(ii) said at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base is selected from the group consisting of three base cutters, four base cutters, five base cutters, and six base cutters, not including degeneracy,

(iii) to produce restriction fragments having N^m different single-stranded overhangs for each restriction endonuclease;

(c) ligating restriction fragments having identical overhangs with a series of adapters whose sequences are complementary to said overhangs;

(d) amplifying said restriction fragments;

(e) separating said amplified restriction fragments to produce a restriction fragment pattern;

(f) obtaining genomic DNA from at least one additional source having a second phenotype or trait of interest;

(g) repeating b) through e) for said DNA from said at least one additional source;

(h) comparing the restriction fragment patterns from each source;

(i) selecting restriction fragments associated with a phenotype or trait of interest; and

(j) using said selected restriction fragments as markers for said phenotype or trait of interest.

2. The method of claim 1, further comprising digesting said genomic DNA with at least one additional restriction endonuclease which is different from the at least one restriction endonuclease of b).

3. The method of claim 2, wherein said additional restriction endonuclease lacks degeneracy in its recognition or cleavage sequence.

4. The method of claim 1, wherein said first phenotype or trait of interest and said second phenotype or trait of interest are the same.

5. The method of claim 1, wherein said first phenotype or trait of interest and said second phenotype or trait of interest are different.

6. The method of claim 1 where N is 2-4.

7. The method of claim 1 where m is 1-5.

8. The method of claim 1, wherein said at least one restriction endonuclease having a degenerate recognition or cleavage sequence is a four base cutter.

9. The method of claim 1, wherein said adaptors comprise a detectable label.

10. The method of claim 1, wherein said amplification incorporates a detectable label into said amplified restriction fragments.

11. The method of claim 1, wherein said amplification is by the polymerase chain reaction.

12. The method of claim 11, wherein primers for said amplification bind to said adapters.

13. The method of claim 12, wherein said primers comprise a detectable label.

14. The method of claim 1, wherein said separation is by electrophoresis.

15. The method of claim 1, further comprising sequencing said amplified sequences.

16. The method of claim 1, further comprising sequencing said amplified sequences selected for use as markers.

17. The method of claim 1, further comprising using said selected markers in marker assisted selection.

18. The method of claim 2, further comprising using said selected markers in marker assisted selection.

19. The method of claim 1, further comprising mapping at least one of said selected markers to a location on a chromosome.

20. The method of claim 2, further comprising mapping at least one of said selected markers to a location on a chromosome.

21. A method for determining DNA methylation patterns comprising:

(a) obtaining genomic DNA from a first source;

(b) digesting said DNA with at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base, wherein;

(i) said degenerate sequence is represented by the formula N^m, where N is the extent of degeneracy, and m is the number of degenerate bases; and

(ii) said at least one restriction endonuclease having a degenerate recognition or cleavage sequence comprising at least one degenerate base is selected from the group consisting of three base cutters, four base cutters, five base cutters, and six base cutters, not including degeneracy,

(iii) to produce restriction fragments having N^m different single-stranded overhangs for each restriction endonuclease;

(c) further digesting said DNA with at least one additional restriction endonuclease that produces a single-stranded overhang, said at least one additional restriction endonuclease being a methylation sensitive endonuclease;

(d) ligating restriction fragments having identical overhangs with a series of adapters whose sequences are complementary to said overhangs;

(e) amplifying said restriction fragments;

(f) separating said amplified restriction fragments to produce a methylation fragment pattern.

22. The method of claim 21 further comprising:

g) obtaining genomic DNA from at least one additional source or from the same source at a different time;

h) repeating b) through e) for said DNA from said at least one additional source;

i) comparing the methylation patterns from each source.