Rapid analysis of variations in a genome

• US 20070196842A1
• Filed: 12/11/2006
• Published: 08/23/2007
• Est. Priority Date: 03/01/2002
• Status: Abandoned Application

• Current Assignee: Ravgen, Inc.
• Original Assignee: Ravgen, Inc.
• Inventors: Dhallan, Ravinder
• Application Number:
  US11/637,354
• Publication Number:
  US 20070196842A1
• Time in Patent Office: Days
• US Class Current: 435/5
• 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 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2545/114 involving a quantitation step

Abstract

The invention provides a method useful for determining the sequence of large numbers of loci of interest on a single or multiple chromosomes. The method utilizes an oligonucleotide primer that contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′ overhang containing the locus of interest. The 5′ overhang is used as a template to incorporate nucleotides, which can be detected. The method is especially amenable to the analysis of large numbers of sequences, such as single nucleotide polymorphisms, from one sample of nucleic acid.

Claims

1. A method for determining a sequence of alleles of a locus of interest, said method comprising:

(a) amplifying alleles of a locus of interest on a template DNA using a first and second primers, wherein the second primer contains a recognition site for a restriction enzyme such that digestion with the restriction enzyme generates a 5′

overhang containing the locus of interest;

(b) digesting the amplified DNA with the restriction enzyme that recognizes the recognition site on the second primer;

(c) incorporating nucleotides into the digested DNA of (b), wherein;

(i) a nucleotide that terminates elongation, and is complementary to the locus of interest of an allele, is incorporated into the 5′

overhang of said allele, and

(ii) a nucleotide complementary to the locus of interest of a different allele is incorporated into the 5′

overhang of said different allele, and said terminating nucleotide, which is complementary to a nucleotide in the 5′

overhang of said different allele, is incorporated into the 5′

overhang of said different allele.

(d) determining the sequence of the alleles of a locus of interest by determining the sequence of the DNA of (c).

2. The method of claim 1, wherein the template DNA is obtained from a source selected from the group consisting of a bacterium, fungus, virus, protozoan, plant, animal and human.

3. The method of claim 1, wherein the template DNA is obtained from a human source.

4. The method of claim 1, wherein the template DNA is obtained from a sample selected from the group consisting of a cell, tissue, blood, serum, plasma, urine, spinal fluid, lymphatic fluid, semen, vaginal secretion, ascitic fluid, saliva, mucosa secretion, peritoneal fluid, fecal matter, or body exudates.

5. The method of claim 1, wherein the amplification in (a) comprises polymerase chain reaction (PCR).

6. The method of claim 1, wherein the restriction enzyme cuts DNA at a distance from the recognition site.

7. The method of claim 1, wherein a 5′

region of the second primer does not anneal to the template DNA.

8. The method of claim 1, wherein a 5′

region of the first primer does not anneal to the template DNA.

9. The method of claim 1, wherein an annealing length of the 3′

region of the second primer is selected from the group consisting of 25-20, 20-15, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, and less than 4 bases.

10. The method of claim 5, wherein an annealing temperature for cycle 1 of PCR is about the melting temperature of the portion of the 3′

region of the second primer that anneals to the template DNA.

11. The method of claim 10, wherein an annealing temperature for cycle 2 of PCR is about the melting temperature of the portion of the 3′

region of the first primer that anneals to the template DNA.

12. The method of claim 11, wherein an annealing temperature for the remaining cycles of PCR is at about the melting temperature of the entire second primer.

13. The method of claim 1, wherein the 3′

end of the second primer is adjacent to the locus of interest.

14. The method of claim 6, wherein the recognition site is for a Type IIS restriction enzyme.

15. The method of claim 14, wherein the Type IIS restriction enzyme is selected from the group consisting of:

Alw I, Alw26 I, Bbs I, Bbv I, BceA I, Bmr I, Bsa I, Bst71 I, BsmA I, BsmB I, BsmF I, BspM I, Ear I, Fau I, Fok I, Hga I, Pie I, Sap I, SSfaN I, and Sthi32 I.

16. The method of claim 14, wherein the Type IIS restriction enzyme is BceA I.

17. The method of claim 14, wherein the Type IIS restriction enzyme is BsmF I.

18. The method of claim 1, wherein the incorporation of a nucleotide in (c) is by a DNA polymerase selected from the group consisting of E. coli DNA polymerase, Klenow fragment of E. coli DNA polymerase I, T7 DNA polymerase, T4 DNA polymerase, Taq polymerase, Pfu DNA polymerase, Vent DNA polymerase and sequenase.

19. The method of claim 1, wherein the incorporation of a nucleotide in (c)(i) comprises incorporation of a labeled nucleotide.

20. The method of claim 1, wherein the incorporation of a nucleotide in (c)(i) comprises incorporation of a dideoxynucleotide.

21. The method of claim 1, wherein the incorporation of a nucleotide in (c)(i) further comprises incorporation of a deoxynucleotide and a dideoxynucleotide.

22. The method of claim 1, wherein the incorporation of a nucleotide in (c)(i) further comprises using a mixture of labeled and unlabeled nucleotides.

23. The method of claim 1, wherein the incorporation of a nucleotide in (c)(ii) comprises incorporation of a labeled nucleotide.

24. The method of claim 1, wherein the incorporation of a nucleotide in (c)(ii) comprises incorporation of a deoxynucleotide.

25. The method of claim 1, wherein the incorporation of a nucleotide in (c)(ii) further comprises incorporation of a deoxynucleotide and a dideoxynucleotide.

26. The method of claim 1, wherein the incorporation of a nucleotide in (c)(ii) further comprises using a mixture of labeled and unlabeled nucleotides.

27. The method of claim 19, wherein the labeled nucleotide is a dideoxynucleotide.

28. The method of claim 19, wherein the labeled nucleotide is labeled with a molecule selected from the group consisting of radioactive molecule, fluorescent molecule, antibody, antibody fragment, hapten, carbohydrate, biotin, derivative of biotin, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, and moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant or electrical conductivity.

29. The method of claim 19, wherein the labeled nucleotide is labeled with a fluorescent molecule.

30. The method of claim 29, wherein the incorporation of a nucleotide in (c)(i) further comprises incorporation of an unlabeled nucleotide.

31. The method of claim 1, wherein the determination of the sequence of the locus of interest in (d) comprises detecting a nucleotide.

32. The method of claim 19, wherein the determination of the sequence of the locus of interest in (d) comprises detecting a labeled nucleotide.

33. The method of claim 32, wherein the detection is by a method selected from the group consisting of gel electrophoresis, polyacrylamide gel electrophoresis, fluorescence detection system, sequencing, ELISA, mass spectrometry, fluorometry, hybridization, microarray, and Southern Blot.

34. The method of claim 32, wherein the detection method is DNA sequencing.

35. The method of claim 32, wherein the detection method is fluorescence detection.

36. The method of claim 1, wherein the alleles of a locus of interest are suspected of containing a single nucleotide polymorphism or mutation.

37. The method of claim 1, wherein the method is used for determining sequences of multiple loci of interest concurrently.

38. The method of claim 37, wherein the template DNA comprises multiple loci from a single chromosome.

39. The method of claim 37, wherein the template DNA comprises multiple loci from different chromosomes.

40. The method of claim 37, wherein the loci of interest on template DNA are amplified in one reaction.

41. The method of claim 37, wherein each of the loci of interest on template DNA is amplified in a separate reaction.

42. The method of claim 41, wherein the amplified DNA are pooled together prior to digestion of the amplified DNA.