Method of detecting genetic polymorphisms using over represented sequences

• US 6,284,466 B1
• Filed: 09/17/1999
• Issued: 09/04/2001
• Est. Priority Date: 09/18/1998
• Status: Expired due to Term

• Current Assignee: University of Nebraska
• Original Assignee: Board of Regents of the University of Nebraska
• Inventors: Benson, Andrew
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/398,499
• Time in Patent Office: 718 Days
• Field of Search: 435/6, 435/91.2, 435/810
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

A method is disclosed for the detection of polymorphisms in a DNA sequence based on nucleotide differences. DNA sequences from different sources are amplified by the polymerase chain reaction using primers based on strand biased, over represented oligonucleotide sequences and differences in the nucleotide sequences of the amplification products determined. Also disclosed are methods for the identification of individuals, or the species, strain or serotype of an organism using the method of the present invention. In addition, kits are disclosed for the determination of polymorphisms using the method of the present invention. The ability to determine genetic polymorphisms has widespread application in areas such as genome mapping, genetic linkage studies, medical diagnosis, epidemiological studies, forensics and agriculture.

Claims

1. A method for determining polymorphism in a DNA sequence on the basis of nucleotide differences comprising,

a) amplifying said DNA sequence to produce amplification products using at least one pair of oligonucleotide primers, said primer pair comprising,

i) a first primer that binds to a first over represented oligonucleotide biased to one strand of said DNA, and

ii) a second primer that binds to a second over represented oligonucleotide biased to a DNA strand complementary to the strand in i),

b) in a separate reaction, repeating a) for at least one other DNA sequence from another source,

c) analyzing the amplification products by a method capable of detecting differences in nucleic acid sequences, and

d) comparing the results obtained for each nucleic acid sequence.

2. The method of claim 1 wherein the DNA is genomic DNA or cDNA.

3. The method of claim 1 wherein the DNA is obtained by reverse transcription of RNA.

4. The method of claim 1 wherein the DNA is obtained from a microorganism.

5. The method of claim 4 wherein the microorganism is E. coli.

6. The method of claim 1 wherein the over represented oligonucleotide is between 6 and 30 bases in length.

7. The method of claim 6 wherein the over represented oligonucleotide is 8 bases in length.

8. The method of claim 4 wherein the primers are selected from the group consisting of Seq. No. 1, Seq. No. 2. Seq. No. 3, Seq. No. 4, Seq. No. 5, Seq. No. 6, Seq. No. 7, Seq. No. 8, Seq. No. 9, Seq. No. 10, Seq. No. 11, Seq. No. 12, Seq. No. 13, Seq. No. 14, Seq. No. 15, Seq. No. 16, Seq. No. 17, Seq. No. 18, Seq. No. 19, Seq. No. 20, Seq. No. 21, Seq. No. 22, Seq. No. 23, Seq. No. 24, Seq. No. 25, Seq. No. 26, Seq. No. 27, Seq. No. 28, Seq. No. 29, Seq. No. 30, Seq. No. 31 Seq. No. 32, Seq. No. 33, Seq. No. 34, Seq. No. 35, Seq. No. 36, Seq. No. 37, Seq. No. 38, Seq. No. 39, Seq. No. 40, Seq. No. 41, Seq. No. 42, Seq. No. 43, Seq. No. 44, Seq. No. 45 and Seq. No. 46.

9. The method of claim 1 further comprising incorporating at least one reporter moiety into the amplification products.

10. The method of claim 9 wherein the reporter moiety is incorporated into at least one primer.

11. The method of claim 9 or 10 wherein the reporter moiety is selected from the group consisting of radionuclides, fluorochromes, peptides, enzymes, antigens, antibodies, vitamins, steroids and dyes.

12. The method of claim 1 wherein the detection of differences in nucleic acid sequences is determined by a method selected from the group consisting of electrophoresis, hybridization, and sequencing.

13. A method for identifying an organism by species, serotype, or strain comprising:

a) obtaining DNA from the organism, said DNA comprising over represented oligonucleotide sequences,

b) amplifying said DNA to produce amplification products using at least one pair of oligonucleotide primers, said primer pair comprising,

i) a first primer that binds to a first over represented oligonucleotide biased to one strand of said DNA, and

ii) a second primer that binds to a second over represented oligonucleotide biased to a DNA strand complementary to the strand in i),

c) analyzing the amplification products by a method capable of detecting differences in nucleic acid sequences, and

d) analyzing the results obtained in c) for amplification products in common with all members of the species and for products unique to strains or serotypes within the species and comparing the results to a database of previously analyzed products, thereby identifying the species, serotype or strain of the organism.

15. The method of claim 13 or 14 wherein the DNA is selected from the group consisting of genomic DNA and cDNA.

16. The method of claim 13 or 14 wherein the DNA is obtained by reverse transcription of RNA into DNA.

17. The method of claim 13 wherein the organism is a microorganism.

18. The method of claim 17 wherein the microorganism is an E. coli.

19. The method of claim 13 or 14 wherein the over represented oligonucleotide is between 6 and 30 bases in length.

20. The method of claim 19 wherein the over represented oligonucleotide is 8 bases in length.

21. The method of claim 17 wherein the primers are selected from the group consisting of Seq. No. 1, Seq. No. 2. Seq. No. 3, Seq. No. 4, Seq. No. 5, Seq. No. 6, Seq. No. 7, Seq. No. 8, Seq. No. 9, Seq. No. 10, Seq. No. 11, Seq. No. 12, Seq. No. 13, Seq. No. 14, Seq. No. 15, Seq. No. 16, Seq. No. 17, Seq. No. 18, Seq. No. 19, Seq. No. 20, Seq. No. 21, Seq. No. 22, Seq. No. 23, Seq. No. 24, Seq. No. 25, Seq. No. 26 Seq. No. 27, Seq. No. 28, Seq. No. 29, Seq. No. 30, Seq. No. 31, Seq. No. 32, Seq. No. 33, Seq. No. 34, Seq. No. 35, Seq. No. 36, Seq. No. 37, Seq. No. 38, Seq. No. 39, Seq. No. 40, Seq. No. 41, Seq. No. 42, Seq. No. 43, Seq. No. 44, Seq. No. 45 and Seq. No. 46.

22. The method of claim 13 or 14 further comprising incorporating at least one reporter moiety into the amplification products.

23. The method of claim 22 wherein the reporter moiety is incorporated into at least one primer.

24. The method of claim 22 wherein the reporter moiety is selected from the group consisting of radionuclides, fluorophores, peptides, enzymes, antigens, antibodies, vitamins, steroids and dyes.

25. The method of claim 13 or 14 wherein the detection of differences in nucleic acid sequences is determined by a method selected from the group consisting of electrophoresis, hybridization, and sequencing.

14. A method for identifying an individual comprising:

a) obtaining DNA from said individual, said DNA comprising over represented oligonucleotide sequences,

b) amplifying said DNA to produce amplification products using at least one pair of oligonucleotide primers, said primer pair comprising,

i) a first primer that binds to a first over represented oligonucleotide biased to one strand of said DNA, and

ii) a second primer that binds to a second over represented oligonucleotide biased to a DNA strand complementary to the strand in i),

c) analyzing the amplification products by a method capable of detecting differences in nucleic acid sequences, and

d) comparing the results obtained in c) to a database of previously analyzed products, thereby identifying the individual.

26. A method for identifying genes by differential display analysis comprising:

a) isolating mRNA from two groups of organisms,

b) converting the mRNA to DNA by reverse transcription,

c) amplifying said DNA to produce amplification products using at least one pair of oligonucleotide primers, said primer pair comprising,

i) a first primer that binds to a first over represented oligonucleotide biased to one strand of said DNA, and

ii) a second primer that binds to a second over represented oligonucleotide biased to a DNA strand complimentary to the strand in i),

d) detecting the amplification products produced by a method capable of detecting the presence of said amplification products, and

e) determining pattern differences in the amplification products produced.

27. The method of claim 26 wherein the organisms are microorganisms.

28. The method of claim 27 wherein the microorganisms are E. coli.

29. The method of claim 26 wherein the over represented oligonucleotide is between 6 and 30 bases in length.

30. The method of claim 29 wherein the over represented oligonucleotide is 8 bases in length.

31. The method of claim 27 wherein the primers are selected from the group consisting of Seq. No. 1, Seq. No. 2. Seq. No. 3, Seq. No. 4, Seq. No. 5, Seq. No. 6, Seq. No. 7, Seq. No. 8, Seq. No. 9, Seq. No. 10, Seq. No. 11, Seq. No. 12, Seq. No. 13, Seq. No. 14, Seq. No. 15, Seq. No. 16, Seq. No. 17, Seq. No. 18, Seq. No. 19, Seq. No. 20, Seq. No. 21, Seq. No. 22, Seq. No. 23, Seq. No. 24, Seq. No. 25, Seq. No. 26, Seq. No. 27, Seq. No. 28, Seq. No. 29, Seq. No. 30, Seq. No. 31 Seq. No. 32, Seq. No. 33, Seq. No. 34, Seq. No. 35, Seq. No. 36, Seq. No. 37, Seq. No. 38, Seq. No. 39, Seq. No. 40, Seq. No. 41, Seq. No. 42, Seq. No. 43, Seq. No. 44, Seq. No. 45 and Seq. No. 46.

32. The method of claim 26 further comprising incorporating at least one reporter moiety into the amplification products.

33. The method of claim 32 wherein the reporter moiety is incorporated into at least one primer.

34. The method of claim 32 or 33 wherein the reporter moiety is selected from the group consisting of radionuclides, fluorophores, peptides, enzymes, antigens, antibodies, vitamins, steroids and dyes.

35. The method of claim 26 wherein the detection of the amplification products produced is by a method selected from the group consisting of electrophoresis and hybridization.

36. The method of claim 26 wherein the detecting method is a quantitative method.

37. The method of claim 36 wherein the quantitative detecting method is selected from the group consisting of densitometry, fluorometry, and radiometry.

38. A kit comprising:

a) at least one pair of oligonucleotide primers, said primer pair comprising,

i) a first primer that binds to a first over represented oligonucleotide biased to one strand of DNA, and

ii) a second primer that binds to a second over represented oligonucleotide biased to a DNA strand complementary to the strand in i),

b) a reaction solution comprising a buffer and a magnesium salt, and

c) instructions for amplification of nucleic acid sequences using the at least one set of primers pairs to produce amplification products and for detection of nucleic acid sequence polymorphism from said amplification products.

39. The kit of claim 38 further comprising dATP, dTTP, dGTP and dCTP.

40. The kit of claim 38 further comprising at least one nucleic acid polymerase.

41. The kit of claim 40 wherein at least one of the at least one nucleic acid polymerase is a thermal stable polymerase.

42. The kit of claim 39 wherein at least one of dATP, dTTP, dGTP and dCTP further comprise a reporter moiety.

43. The kit of claim 38 wherein at least one member of the at least one primer pair further comprises a reporter moiety.

44. The kit of claim 42 or 43 wherein the reporter moiety is selected from the group consisting of radionuclides, fluorophores, peptides, enzymes, antigens, antibodies, vitamins, steroids and dyes.

45. A method for the determination of polymorphism in a DNA sequence comprising:

a) producing an oligonucleotide sequence containing a reporter moiety, said oligonucleotide comprising an over represented oligonucleotide,

b) if desired, repeating step a) at least once to produce at least one additional oligonucleotide sequence containing a reporter moiety, said additional oligonucleotide sequence comprising a different over represented oligonucleotide,

c) contacting the oligonucleotide of a) and if present b) with chromosomes obtained from an organism, so that the oligonucleotide hybridizes to complementary nucleotide sequences contained in said chromosomes,

d) repeating c) with chromosomes obtained from a different organism,

e) detecting the presence of the oligonucletides on the chromosomes, and

f) comparing the locations of the oligonucleotides on the chromosomes to detect polymorphism.

46. The method of claim 45 wherein the polymorphism detected is used to identify individuals.

47. The method of claim 45 wherein the reporter moiety is selected from the group consisting of radionuclides, fluorochromes, peptides, enzymes, antigens, antibodies, vitamins, steroids and dyes.

48. The method of claim 45 wherein the reporter moiety is a fluorochrome.

49. The method of claim 45 wherein the location of the oligonucleotide is detected by microscopy.

50. The method of claim 49 wherein the location of the oligonucleotide is detected by fluorescence microscopy.

51. The method of claim 49 wherein the location of the oligonucleotide is detected by confocal microscopy.