Methods for identifying a mutation in a gene of interest without a phenotypic guide

• US 5,994,075 A
• Filed: 05/16/1997
• Issued: 11/30/1999
• Est. Priority Date: 05/17/1996
• Status: Expired due to Fees

• Current Assignee: Ingenium Pharmaceuticals AG
• Original Assignee: Hexagen Technology Ltd (Incyte Corporation)
• Inventors: Goodfellow, Peter N.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US08/857,946
• Time in Patent Office: 928 Days
• Field of Search: 435/6, 435/91.2, 435/441, 435/444, 435/446
• US Class Current: 435/6.16
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2565/131 Single/double strand conformational analysis, i.e. SSCP/DSCP

Abstract

The invention encompasses methods of identifying a mutation in a gene of interest in an organism which include identifying in a test DNA sample from a mutated organism a mutation in a gene of interest without the prior observation of a phenotypic alteration in the mutated organism.

Claims

1. A method of identifying a mutation in a gene of interest in an organism, comprising

mutagenizing a non-transgenic organism to produce a mutated organism;

and

testing a DNA sample from said mutated organism for a mutation in a gene of interest without the prior observation of a phenotypic alteration in said mutated organism.

3. The method of claim 1 or 2 wherein said mutated organism is the same as said organism which is mutagenized.

4. The method of claim 1 or 2 wherein said mutated organism is the offspring of said organism which is mutagenized.

5. The method of claim 4 wherein said offspring is the F1 generation of said organism which is mutagenized.

6. The method of claim 4 wherein said offspring is the F2 or a subsequent generation of said organism which is mutagenized.

7. The method of claim 1 or 2 wherein said testing comprises hybridization of a DNA probe to said sample or said mixture, said probe being unique to said gene of interest.

8. The method of claim 1 or 2 wherein said testing comprises hybridization of a mixture of multiple DNA probes to said sample or said mixture, said multiple DNA probes differing in sequence from each other and being unique to said gene of interest.

14. The method of claim 1 wherein said mutated organism contains about 1 mutation in 10,000 genes-1 mutation in 1000 genes.

17. The method of any of claims 1, 2, 9, 13 or 16 wherein said mutagenizing step comprises inducing a genetic mutation into an organism at an average frequency of 1/1000-1/10,000 organisms.

18. The method of claim 1 wherein the mutation is a single base pair mutation.

19. The method of claim 1 wherein the mutation is an insertion or deletion mutation.

20. The method of claim 1, 2, 9, 13, or 17 wherein said mutagenizing step comprises exposing said organism to an alkylating agent.

21. The method of claim 20 wherein said alkylating agent comprises ENU or MNU.

22. The method of claim 1, 2, 9, 13, or 16 wherein said mutagenizing step comprises mutating germline DNA of said organism.

23. The method of claim 7 wherein said probe or probes comprises a pair of unique PCR primers.

24. The method of claim 23 wherein said testing comprises amplification of a segment of said gene of interest and sequencing of said amplified segment.

25. The method of claim 24 wherein said testing comprises fSSCP analysis.

27. The method of claim 1 wherein said organism is a mammal.

28. The method of claim 27, said mammal being a mouse.

50. The method of claim 8 wherein said probe or probes comprises a pair of unique PCR primers.

52. The method of claim 50, wherein said testing comprises amplification of a segment of said gene of interest and sequencing of said amplified segment.

2. A method of identifying a mutation in a gene of interest in an organism, comprising

mutagenizing a plurality of the same non-transgenic organism to produce mutated organisms;

and

testing a mixture of pooled DNA samples containing a plurality of DNA samples from a corresponding plurality of mutated organisms for a mutation in a gene of interest without the prior observation of a phenotypic alteration in said mutated organisms.

9. A method of identifying a mutation in a gene of interest in an organism, comprising

mutagenizing the germline DNA of a non-transgenic organism;

mating said mutagenized organism to produce F1 offspring; and

testing a DNA sample from a said F1 offspring for a mutation in said gene of interest without the prior observation of a phenotypic alteration in said F1 offspring.

10. The method of claim 9 wherein said germline DNA is from a male organism.

11. A method of identifying a mutation in a gene of interest of an organism, the method comprising the steps of

a) providing a mixture of pooled DNA samples, each said DNA sample of said pool being from a mutated organism;

b) providing a nucleic acid probe unique to a gene of interest;

c) testing said mixture for a mutation in said gene of interest by hybridizing said probe to said mixture without the prior observation of a phenotypic alteration in each said mutated organism.

12. The method of claim 11, further comprising the steps of

d) detecting a said mutation in said mixture;

and

e) testing each said DNA sample individually for a mutation in said gene of interest.

13. The method of claim 11, further comprising, prior to step a), the step of

mutagenizing a plurality of the same organism.

26. The method of claim 2 or 13 wherein said plural mutated organisms comprise about 1,000-10,000 organisms.

51. The method of claim 11, or 15 wherein said probe or probes comprises a pair of unique PCR primers.

53. The method of claim 51, wherein said testing comprises amplification of a segment of said gene of interest and sequencing of said amplified segment.

15. A method of identifying a mutation in two or more genes of interest in an organism, the method comprising the steps of

a) providing a DNA sample from a given tissue of a mutated non-transgenic organism, wherein said mutated organism contains about 1 mutation in 1000 genes,

b) providing plural nucleic acid probes, each said probe being unique to a given gene of interest,

c) testing said DNA sample for a mutation in a said gene of interest without the prior observation of a phenotypic alteration in said mutated organism.

16. The method of claim 15, further comprising prior to step a) the step of

mutagenizing an organism to produce said mutated organism.

29. In a method of inducing and identifying a mutation in a gene of interest in a non-transgenic organism, wherein the organism is exposed to a mutagen so as to induce a mutation in the genome of the organism, and the organism is then mated to produce an offspring containing the gene of interest, the improvement comprising the step of

testing the gene of interest for a mutation without the prior selection of a phenotypic characteristic in the offspring.

30. The method of claim 29 wherein said improvement comprises testing the gene of interest for a mutation by hybridization of a DNA probe to a DNA sample of said offspring, said probe being unique to said gene of interest.

31. The method of claim 30 wherein said improvement comprises testing the gene of interest for a mutation using a DNA probe which comprises a mixture of multiple DNA probes differing in sequence from each other and being unique to said gene of interest.

32. The method of claim 29 wherein in said method which is improved, the induced mutation is a single base pair mutation.

33. The method of claim 29 wherein in said method which is improved, the induced mutation is an insertion or deletion mutation.

34. The method of claim 29 wherein in said method which is improved, the mutagen comprises an alkylating agent.

35. The method of claim 34 wherein said alkylating agent comprises ENU or MNU.

36. The method of claim 29 wherein in said method improved, the organism is exposed to a mutagen by mutating germline DNA of said organism.

37. The method of claim 30 wherein said DNA probe comprises a pair of unique PCR primers.

38. The method of claim 37 wherein said testing comprises amplification of a segment of said gene of interest and sequencing of said amplified segment.

39. The method of claim 38 wherein said testing comprises fSSCP analysis.

40. The method of claim 29, wherein in the method which is improved said organism is a mammal.

41. The method of claim 40, said mammal being a mouse.

42. A method of identifying a mutation in a gene of interest in an organism, consisting essentially of:

a) mutagenizing a non-transgenic organism so as to produce a mutated organism; and

b) testing a DNA sample from said mutated organism for a mutation in a gene of interest without the prior observation of a phenotypic alteration in said mutated organism.

43. A method of identifying a mutation in a gene of interest in an organism, consisting essentially of:

a) mutagenizing a plurality of the same non-transgenic organism to produce mutated organisms; and

b) testing a mixture of pooled DNA samples containing a plurality of DNA samples from a corresponding plurality of mutated organisms for a mutation in a gene of interest without the prior observation of a phenotypic alteration in said mutated organisms.

44. A method of identifying a mutation in a gene of interest in an organism, consisting essentially of:

a) mutagenizing the germline DNA of a non-transgenic organism;

b) mating said mutagenized organism to produce F1 offspring; and

c) testing a DNA sample from a said F1 offspring for a mutation in said gene of interest without the prior observation of a phenotypic alteration in said F1 offspring.

45. A method of identifying a mutation in a gene of interest of an organism, the method consisting essentially of:

a) providing a mixture of pooled DNA samples, each said DNA sample of said pool being from a mutated non-transgenic organism;

b) providing a nucleic acid probe unique to a gene of interest; and

c) testing said mixture for a mutation in said gene of interest by hybridizing said probe to said mixture without the prior observation of a phenotypic alteration in each said mutated organism.

46. The method of claim 45, further consisting essentially of:

d) detecting a said mutation in said mixture; and

e) testing each said DNA sample individually for a mutation in said gene of interest.

47. The method of claim 45, further consisting essentially of, prior to step a), the step of

mutagenizing a plurality of the same organism.

48. A method of identifying a mutation in two or more genes of interest in an organism, the method consisting essentially of:

a) providing a DNA sample from a given tissue of a mutated non-transgenic organism, wherein said mutated organism contains about 1 mutation in 1000 genes,

b) providing plural nucleic acid probes, each said probe being unique to a given gene of interest, and

c) testing said DNA sample for a mutation in a said gene of interest without the prior observation of a phenotypic alteration in said mutated organism.

49. The method of claim 48, further consisting essentially of, prior to step a) the step of

mutagenizing an organism to produce said mutated organism.