Rapid analysis of variations in a genome

• US 6,977,162 B2
• Filed: 03/11/2002
• Issued: 12/20/2005
• Est. Priority Date: 03/01/2002
• Status: Expired due to Term

• Current Assignee: Ravgen, Inc.
• Original Assignee: Ravgen, Inc.
• Inventors: Dhallan, Ravinder S.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Kim, Young J.
• Application Number:
  US10/093,618
• Publication Number:
  US 20030186239A1
• Time in Patent Office: 1,380 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/24.3, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• 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 1/6869 Methods for sequencing
• 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 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/113 PCR
• 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
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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 a locus of interest, said method comprising:

(a) amplifying a locus of interest on a template DNA using a first and second primers, each of the first and second primers comprising a 3′

annealing region that anneals to the template DNA, 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, and wherein the melting temperature of the 3′

annealing region of the first primer is greater than the melting temperature of the 3′

annealing region of the second primer, and further wherein the annealing temperature for cycle 1 of the amplification is at about the melting temperature of the 3′

annealing region of the second primer, the annealing temperature for cycle 2 of the amplification is at about the melting temperature of the 3′

annealing region of the first primer, and the annealing temperature for cycle 3 is at about the melting temperature of the entire second primer, and wherein the annealing temperatures for cycle 2 and cycle 3 are greater than the annealing temperature for cycle 1;

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

(c) incorporating a nucleotide into the digested DNA of (b) by using the 5′

overhang containing the locus of interest as a template; and

(d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c) containing the incorporated nucleotide.

2. The method of claim 1, wherein the amplification of step (a) comprises cycles of amplification subsequent to cycle 3, wherein the annealing temperature of the cycles of amplification subsequent to cycle 3 is at about the melting temperature of the entire second primer.

3. 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.

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

5. 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, and body exudates.

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

7. The method of claim 1, wherein the restriction enzyme cuts DNA at the recognition site.

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

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

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

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

10. The method of claim 7, wherein the restriction enzyme is selected from the group consisting of BsaJ I, Bssk I, Dde I, EcoN I, Fnu4H I, and Hinf I.

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

12. The method of claim 11, wherein a 5′

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

13. The method of claim 11, wherein a 5′

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

14. The method of claim 11, 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.

15. The method of claim 11, wherein the recognition site is for a Type IIS restriction enzyme.

16. The method of claim 15, 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, Ple I, Sap I, SSfaN I, and Sthi32 I.

17. The method of claim 15, wherein the Type IIS restriction enzyme is BceA I or BsmF I.

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

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

19. The method of claim 1, wherein the first primer contains a recognition site for a restriction enzyme that is different from the recognition site for the restriction enzyme on the second primer.

20. The method of claim 19, further comprising digesting the DNA of (c) with a restriction enzyme that recognizes the recognition site on the first primer.

21. The method of claim 1, wherein the first primer, the second primer, or both the first primer and the second primer contain a tag at the 5′

terminus.

22. The method of claim 1, wherein the first primer contains a tag at the 5′

terminus.

23. The method of claim 22, wherein the tag is used to separate the amplified DNA from the template DNA.

24. The method of claim 23, wherein the tag is used to separate the amplified DNA containing the incorporated nucleotide from the amplified DNA that does not contain the incorporated nucleotide.

25. The method of claim 22, wherein the tag is selected from the group consisting of:

radioisotope, fluorescent reporter molecule, chemiluminescent reporter molecule, antibody, antibody fragment, hapten, biotin, derivative of biotin, photobiotin, iminobiotin, digoxigenin, avidin, enzyme, acridinium, sugar, enzyme, apoenzyme, homopolymeric oligonucleotide, hormone, ferromagnetic moiety, paramagnetic moiety, diamagnetic moiety, phosphorescent moiety, luminescent moiety, electrochemiluminescent moiety, chromatic moiety, moiety having a detectable electron spin resonance, electrical capacitance, dielectric constant, electrical conductivity, and combinations thereof.

26. The method of claim 22, wherein the tag is biotin.

27. The method of claim 26, wherein the biotin tag is used to separate amplified DNA from the template DNA using a streptavidin matrix.

28. The method of claim 27, wherein the streptavidin matrix is coated on wells of a microtiter plate.

29. 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.

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

31. The method of claim 30, wherein the labeled nucleotide is selected from the group consisting of a dideoxynucleotide triphosphate and deoxynucleotide triphosphate.

32. The method of claim 30, 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, moiety having a detectable electron spin resonance, moiety having an electrical capacitance, moiety having a dielectric constant, and moiety having a detectable electrical conductivity.

33. The method of claim 30, wherein the labeled nucleotide is labeled with a fluorescent molecule.

34. The method of claim 33, wherein the incorporation of a fluorescent labeled nucleotide further comprises using a mixture of fluorescent and unlabeled nucleotides.

35. The method of claim 30, wherein the determination of the sequence of the locus of interest in (d) comprises detecting labeled nucleotide.

36. The method of claim 35, 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.

37. The method of claim 35, wherein the detection method is DNA sequencing.

38. The method of claim 35, wherein the detection method is a fluorescence detection system.

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

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

41. The method of claim 1, wherein the locus of interest is suspected of containing a single nucleotide polymorphism or mutation.

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

43. The method of claim 42, wherein the template DNA comprises multiple loci from a single chromosome.

44. The method of claim 42, wherein the template DNA comprises multiple loci from different chromosomes.

45. The method of claim 42, wherein the loci of interest on template DNA are amplified in one reaction.

46. The method of claim 42, wherein each of the loci of interest on template DNA is amplified in a separate reaction.

47. The method of claim 46, wherein the amplified DNA are pooled together prior to digestion of the amplified DNA.

48. The method of claim 42, wherein each of the DNA of (c) containing the incorporated nucleotide and containing a locus of interest is separated from other loci of interest prior to (d).

49. The method of claim 42, wherein at least one of the loci of interest is suspected of containing a single nucleotide polymorphism or a mutation.

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

(a) amplifying a locus of interest on a template DNA using a first and second primers, each of the first and second primers comprising a 3′

annealing region that anneals to the template DNA, 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, and wherein the melting temperature of the 3′

annealing region of the first primer is greater than the melting temperature of the 3′

annealing region of the second primer, and further wherein the annealing temperature for cycle 1 of the amplification is at about the melting temperature of the 3′

annealing region of the second primer, and the annealing temperature for a cycle of amplification subsequent to the first cycle of amplification is at about the melting temperature of the 3′

annealing region of the first primer, and wherein the annealing temperature for said cycle of amplification subsequent to the first cycle of amplification is greater than the annealing temperature for cycle 1;

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

(c) incorporating a nucleotide into the digested DNA of (b) by using the 5′

overhang containing the locus of interest as a template; and

(d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c) containing the incorporated nucleotide.

51. The method of claim 50, wherein the 5′

region of the second primer does not anneal to the template DNA, and further wherein in a cycle of amplification subsequent to the cycle of amplification for which the annealing temperature is at about the melting temperature of the 3′

annealing region of the first primer, the annealing temperature is at about the melting temperature of the entire second primer.

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

(a) amplifying a locus of interest on a template DNA using a first and second primers, each of the first and second primers comprising a 3′

annealing region that anneals to the template DNA, wherein the second primer contains a recognition site for a restriction enzyme that cuts DNA at a distance from the recognition site and digestion with the restriction enzyme generates a 5′

overhang containing the locus of interest, wherein the first primer contains a recognition site for a restriction enzyme that is different from the recognition site for the restriction enzyme on the second primer, and contains a tag at the 5′

end, and wherein the melting temperature of the 3′

annealing region of the first primer is greater than the melting temperature of the 3′

annealing region of the second primer, and further wherein the annealing temperature for cycle 1 of the amplification is at about the melting temperature of the 3′

annealing region of the second primer, the annealing temperature for cycle 2 of the amplification is at about the melting temperature of the 3′

annealing region of the first primer, and the annealing temperature for cycle 3 is at about the melting temperature of the entire second primer, and further wherein the annealing temperatures for cycle 2 and cycle 3 are greater than the annealing temperature for cycle 1;

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

(c) incorporating a labeled nucleotide into the digested DNA of (b) by using the 5′

overhang containing the locus of interest as a template;

(d) digesting the DNA of (c) with the restriction enzyme that recognizes the recognition site on the first primer; and

(e) determining the sequence of the locus of interest by determining the sequence of the digested DNA of (d) containing the labeled nucleotide.

53. The method of claim 52, wherein the tag is used to separate the amplified DNA from the template DNA.

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

(a) amplifying a locus of interest on a template DNA using a first and second primers, each of the first and second primers comprising a 3′

annealing region that anneals to the template DNA, 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, wherein the 5′

region of the second primer does not anneal to the template DNA, and wherein the annealing temperature for cycle 1 of the amplification is at about the melting temperature of the 3′

annealing region of the second primer, and the annealing temperature for a cycle of amplification subsequent to the first cycle of amplification is at about the melting temperature of the entire second primer, and wherein the annealing temperature for said cycle of amplification subsequent to the first cycle of amplification is greater than the annealing temperature for cycle 1;

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

(c) incorporating a nucleotide into the digested DNA of (b) by using the 5′

overhang containing the locus of interest as a template; and

(d) determining the sequence of the locus of interest by determining the sequence of the DNA of (c) containing the incorporated nucleotide.