Method for identifying genes underlying defined phenotypes

• US 6,221,585 B1
• Filed: 01/15/1998
• Issued: 04/24/2001
• Est. Priority Date: 01/15/1998
• Status: Expired due to Fees

• Current Assignee: Valigen (US), Inc.
• Original Assignee: Valigen (US), Inc.
• Inventors: Iris, Francois J. -M., Pourny, Jean-Louis
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/007,905
• Time in Patent Office: 1,195 Days
• Field of Search: 435/6, 435/91.2, 435/7.1, 435/513
• US Class Current: 435/6.16
• 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 2522/10 Nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2563/119 the label being proteinic

Abstract

The present invention relates generally to the field of genomics. More particularly, the present invention relates to a method for gene identification beginning with user-selected input phenotypes. The method is referred to generally as the ValiGene^SMGene Identification method, or the VGID^SM method. The method employs nucleic acid mismatch binding protein chromatography to effect a molecular comparison of one phenotype with others. Genes are identified as having a specified function, or as causing or contributing to the cause or pathogenesis of a specified disease, or as associated with a specific phenotype, by virtue of their selection by the method. Identified genes may be used in development of reagents, drugs and/or combinations thereof useful in clinical or other settings for prognosis, diagnosis and/or treatment of diseases, disorders and/or conditions. The method is equally suited for gene identification for agricultural, bio-engineering, medical, veterinary, and many other applications.

Claims

1. A method for identifying one or more genes underlying a defined phenotype comprising the following steps in the order stated:

(a) removing mismatched duplex nucleic acid molecules formed from hybridization within each of two source populations of nucleic acids; and

(b) retaining mismatched duplex nucleic acid molecules formed from hybridization between the two source populations,

the retained molecules in step (b) comprising the one or more genes underlying the defined phenotype.

5. The method of claim 1 or claim 2, wherein the source populations are normalized cDNA libraries.

6. The method of claim 1 or claim 2, wherein the source populations are linearized cDNA libraries.

7. The method of claim 1, wherein the two source populations are of DNA, the DNA of the second source population is labeled, and the hybridization in step (b) is carried out using an excess of labeled DNA.

8. The method of claim 7, wherein the excess of labeled DNA is a three-fold excess.

12. The method of claim 1, wherein the two source populations are each derived from a cell line.

30. The method of claim 1 or claim 2, wherein the defined phenotype is selected from the group consisting of a plant phenotype, a microorganism phenotype, and a pathologic phenotype.

31. The method of claim 30, wherein the pathologic phenotype is selected from the group consisting of cancer, osteoporosis, obesity, type II diabetes, and a prion-related disease.

55. The method of any one of claims 1, 2, 13, 14, 32-35, 51 and 53, wherein the one or more identified genes or alleles is used to carry out a prognosis or a diagnosis.

2. A method for identifying one or more genes underlying a defined phenotype comprising the following steps in the order stated:

(a) removing mismatched duplex nucleic acid molecules formed from hybridization within a first source population of nucleic acids; and

(b) retaining mismatched duplex nucleic acid molecules formed from hybridization between the first source population and a second source population of nucleic acids,

the retained molecules in step (b) comprising the one or more genes underlying the defined phenotype.

3. The method of claim 2, wherein the first and second source populations each are nucleic acid populations derived from at least two individuals having consanguinity.

4. The method of claim 2, wherein the first and second source populations each are nucleic acid populations derived from more than two individuals having consanguinity.

9. The method of claim 2, wherein the two source populations are of DNA, the DNA of the first source population is labeled, and the hybridization in step (b) is carried out using an excess of labeled DNA.

10. The method of claim 9, wherein the excess of labeled DNA is a three-fold excess.

11. The method of any one of claims 7-10, wherein labeling of DNA is carried out by polymerase chain reaction using a 5′

-biotinylated primer.

13. A method for identifying one or more genes underlying a defined phenotype displayed by a cell or individual from which a first cDNA library is derived, but not displayed by a cell or individual from which a second cDNA library is derived, comprising:

(a) hybridizing insert DNA from the first cDNA library with itself;

(b) hybridizing insert DNA from the second cDNA library with itself;

(c) contacting the DNA hybridized in step (a) with a first immobilized mismatch binding protein;

(d) contacting the DNA hybridized in step (b) with a second immobilized mismatch binding protein;

(e) separating unbound DNA from bound DNA contacted in step (c);

(f) separating unbound DNA from bound DNA contacted in step (d);

(g) labeling unbound DNA separated in step (f) with a label capable of binding a partner molecule immobilized on a substrate;

(h) hybridizing DNA labeled in step (g) with unbound DNA separated in step (e);

(i) contacting DNA hybridized in step (h) with a third immobilized mismatch binding protein;

(j) separating unbound DNA from bound DNA contacted in step (i);

(k) contacting unbound DNA separated in step (j) with the partner molecule immobilized on the substrate capable of binding the label; and

(l) separating unbound DNA from bound DNA contacted in step (k),

which unbound DNA separated in step (l) encodes one or more identified genes underlying the defined phenotype.

17. The method of claim 13 or claim 14, wherein the cDNA libraries are normalized.

18. The method of claim 13 or claim 14, wherein the cDNA libraries are linearized.

19. The method of claim 13 or claim 14, wherein labeling is carried out by polymerase chain reaction using a 5′

-biotinylated primer.

20. The method of claim 13 or claim 14, wherein labeling is carried out by polymerase chain reaction using a 5′

-peptide labeled primer.

21. The method of claim 13 or claim 14, wherein the immobilized partner molecule is streptavidin.

22. The method of claim 13 or claim 14, wherein the immobilized partner molecule is an antibody.

23. The method of claim 22, wherein the antibody is an anti-peptide antibody.

24. The method of claim 13, wherein the hybridization in step (h) is carried out using an excess of labeled DNA.

25. The method of claim 24, wherein the excess of labeled DNA is a three-fold excess.

28. The method of claim 13, wherein the first, second and third immobilized mismatch binding proteins are MutS.

14. A method for identifying one or more genes underlying a defined phenotype from organisms having consanguinity comprising:

(a) hybridizing insert DNA from a first collection of cDNA libraries derived from organisms having the defined phenotype with itself;

(b) contacting the DNA hybridized in step (a) with a first immobilized mismatch binding protein;

(c) separating unbound DNA from bound DNA contacted in step (b);

(d) labeling unbound DNA separated in step (c) with a label capable of binding a partner molecule immobilized on a substrate;

(e) hybridizing DNA labeled in step (d) with insert DNA from a second collection of cDNA libraries derived from organisms not having the defined phenotype;

(f) contacting DNA hybridized in step (e) with a second immobilized mismatch binding protein;

(g) separating unbound DNA from bound DNA contacted in step (f);

(h) contacting unbound DNA separated in step (g) with the partner molecule immobilized on the substrate capable of binding the label; and

(i) separating unbound DNA from bound DNA contacted in step (h),

which unbound DNA separated in step (i) encodes one or more dentified genes underlying the defined phenotype.

15. The method of claim 14, wherein the first and second cDNA library collections each are nucleic acid populations derived from at least two individuals having consanguinity.

16. The method of claim 14, wherein the first and second cDNA library collections each are nucleic acid populations derived from more than two individuals having consanguinity.

26. The method of claim 14, wherein the hybridization in step (e) is carried out using an excess of labeled DNA.

27. The method of claim 26, wherein the excess of labeled DNA is a three-fold excess.

29. The method of claim 14, wherein the first and second immobilized mismatch binding proteins are MutS.

32. A method for identifying one or more genes underlying a defined phenotype displayed by a cell or individual from which a first cDNA library is derived, but not displayed by a cell or individual from which a second cDNA library is derived, comprising:

(a) amplifying insert DNA from the first cDNA library by polymerase chain reaction;

(b) amplifying insert DNA from the second cDNA library by polymerase chain reaction;

(c) hybridizing DNA amplified in step (a) with itself;

(d) hybridizing DNA amplified in step (b) with itself;

(e) contacting DNA hybridized in step (c) with a first immobilized MutS;

(f) contacting DNA hybridized in step (d) with a second immobilized MutS;

(g) separating unbound DNA from bound DNA contacted in step (e);

(h) separating unbound DNA from bound DNA contacted in step (f);

(i) amplifying unbound DNA separated in step (g) by polymerase chain reaction using unlabeled primers;

(j) amplifying and labeling unbound DNA separated in step (h) by polymerase chain reaction using 5′

-biotinylated primers;

(k) hybridizing DNA amplified and labeled in step (j) with DNA amplified in step (i);

(l) contacting DNA hybridized in step (k) with a third immobilized MutS;

(m) separating unbound DNA from bound DNA contacted in step (l);

(n) contacting unbound DNA separated in step (m) with immobilized streptavidin; and

(o) separating unbound DNA from bound DNA contacted in step (n),

which unbound DNA separated in step (o) encodes one or more identified genes underlying the defined phenotype.

33. A method for identifying one or more genes underlying a disease phenotype from healthy and affected individuals having consanguinity comprising:

(a) amplifying insert DNA from a first collection of cDNA libraries derived from affected individuals by polymerase chain reaction;

(b) hybridizing DNA amplified in step (a) with itself;

(c) contacting DNA hybridized in step (b) with a first immobilized MutS;

(d) separating unbound DNA from bound DNA contacted in step (c);

(e) amplifying and labeling unbound DNA separated in step (d) by polymerase chain reaction using 5′

-biotinylated primers;

(f) amplifying insert DNA from a second collection of cDNA libraries derived from healthy individuals by polymerase chain reaction;

(g) hybridizing DNA amplified and labeled in step (e) with DNA amplified in step (f);

(h) contacting DNA hybridized in step (g) with a second immobilized MutS;

(i) separating unbound DNA from bound DNA contacted in step (h);

(j) contacting unbound DNA separated in step (i) with immobilized streptavidin; and

(k) separating unbound DNA from bound DNA contacted in step (j),

which unbound DNA separated in step (k) encodes one or more identified genes underlying the disease phenotype.

34. A method for identifying one or more alleles underlying a defined phenotype displayed by a cell or individual from which a first cDNA library is derived, but not displayed by a cell or individual from which a second cDNA library is derived, comprising:

(a) hybridizing insert DNA from the first cDNA library with itself;

(b) hybridizing insert DNA from the second cDNA library with itself;

(c) contacting DNA hybridized in step (a) with a first immobilized mismatch binding protein;

(d) contacting DNA hybridized in step (b) with a second immobilized mismatch binding protein;

(e) separating unbound DNA from bound DNA contacted in step (c);

(f) separating unbound DNA from bound DNA contacted in step (d);

(g) labeling unbound DNA separated in step (f) with a label capable of binding a partner molecule immobilized on a substrate;

(h) hybridizing DNA labeled in step (g) with unbound DNA separated in step (e);

(i) contacting DNA hybridized in step (h) with a third immobilized mismatch binding protein;

(j) separating unbound DNA from bound DNA contacted in step (i);

(k) releasing bound DNA separated in step (j) from the third immobilized mismatch binding protein;

(l) contacting DNA released in step (k) with the partner molecule immobilized on the substrate capable of binding the label;

(m) denaturing DNA contacted in step (l); and

(n) separating unbound DNA from bound DNA denatured in step (m),

which unbound DNA separated in step (n) encodes one or more identified alleles underlying the defined phenotype.

38. The method of claim 34 or claim 35, wherein the cDNA libraries are normalized.

39. The method of claim 34 or claim 35, wherein the cDNA libraries are linearized.

40. The method of claim 34 or claim 35, wherein labeling is carried out by polymerase chain reaction using a 5′

-biotinylated primer.

41. The method of claim 34 or claim 35, wherein labeling is carried out by polymerase chain reaction using a 5′

-peptide labeled primer.

42. The method of claim 34 or claim 35, wherein the immobilized partner molecule is streptavidin.

43. The method of claim 34 or claim 35, wherein the immobilized partner molecule is an antibody.

44. The method of claim 43, wherein the antibody is an anti-peptide antibody.

45. The method of claim 34, wherein the hybridization in step (h) is carried out using an excess of labeled DNA.

46. The method of claim 45, wherein the excess of labeled DNA is a three-fold excess.

49. The method of claim 34, wherein the first, second and third immobilized mismatch binding proteins are MutS.

35. A method for identifying one or more alleles underlying a defined phenotype from organisms having consanguinity comprising:

(a) hybridizing insert DNA from a first collection of cDNA libraries derived from organisms having the defined phenotype with itself;

(b) contacting DNA hybridized in step (a) with a first immobilized mismatch binding protein;

(c) separating unbound DNA from bound DNA contacted in step (b);

(d) labeling unbound DNA separated in step (c) with a label capable of binding a partner molecule immobilized on a substrate;

(e) hybridizing DNA labeled in step (d) with insert DNA from a second collection of cDNA libraries derived from organisms not having the defined phenotype;

(f) contacting DNA hybridized in step (e) with a second immobilized mismatch binding protein;

(g) separating unbound DNA from bound DNA contacted in step (f);

(h) releasing bound DNA separated in step (g) from the second immobilized mismatch binding protein;

(i) contacting DNA released in step (h) with the partner molecule immobilized on the substrate capable of binding the label;

(j) denaturing DNA contacted in step (i); and

(k) separating bound DNA from unbound DNA denatured in step (j),

which bound DNA separated in step (k) encodes one or more identified alleles underlying the defined phenotype.

36. The method of claim 35, wherein the first and second cDNA library collections each are nucleic acid populations derived from at least two individuals having consanguinity.

37. The method of claim 35, wherein the first and second cDNA library collections each are nucleic acid populations derived from more than two individuals having consanguinity.

47. The method of claim 35, wherein the hybridization in step (e) is carried out using an excess of labeled DNA.

48. The method of claim 47, wherein the excess of labeled DNA is a three-fold excess.

50. The method of claim 35, wherein the first and second immobilized mismatch binding proteins are MutS.

51. A method for identifying one or more alleles underlying a defined phenotype displayed by a cell or individual from which a first cDNA library is derived, but not displayed by a cell or individual from which a second cDNA library is derived, comprising:

(a) amplifying insert DNA from the first cDNA library by polymerase chain reaction;

(b) amplifying insert DNA from the second cDNA library by polymerase chain reaction;

(c) hybridizing DNA amplified in step (a) with itself;

(d) hybridizing DNA amplified in step (b) with itself;

(e) contacting DNA hybridized in step (c) with a first immobilized MutS;

(f) contacting DNA hybridized in step (d) with a second immobilized MutS;

(g) separating unbound DNA from bound DNA contacted in step (e);

(h) separating unbound DNA from bound DNA contacted in step (f);

(i) amplifying unbound DNA separated in step (g) by polymerase chain reaction using unlabeled primers;

(j) amplifying and labeling unbound DNA separated in step (h) by polymerase chain reaction using 5′

-biotinylated primers;

(k) hybridizing DNA amplified and labeled in step (j) with DNA amplified in step (i);

(l) contacting DNA hybridized in step (k) with a third immobilized MutS;

(m) separating unbound DNA from bound DNA contacted in step (l);

(n) releasing bound DNA separated in step (m) from the third immobilized MutS;

(o) contacting DNA released in step (n) with immobilized streptavidin;

(p) denaturing DNA contacted in step (o);

(q) separating unbound DNA from bound DNA denatured in step (p),

which unbound DNA separated in step (q) encodes one or more identified alleles underlying the defined phenotype.

52. The method of claim 51, wherein releasing bound DNA from the third immobilized MutS in step (n) is carried out using ATP or proteinase K.

53. A method for identifying one or more affected alleles underlying a disease phenotype from healthy and affected individuals having consanguinity comprising:

(a) amplifying insert DNA from a first collection of cDNA libraries derived from affected individuals by polymerase chain reaction;

(b) hybridizing DNA amplified in step (a) with itself;

(c) contacting DNA hybridized in step (b) with a first immobilized MutS;

(d) separating unbound DNA from bound DNA contacted in step (c);

(e) amplifying and labeling unbound DNA separated in step (d) by polymerase chain reaction using 5′

-biotinylated primers;

(f) amplifying insert DNA from a second collection of cDNA libraries derived from healthy individuals by polymerase chain reaction;

(g) hybridizing DNA amplified and labeled in step (e) with DNA amplified in step (f);

(h) contacting DNA hybridized in step (g) with a second immobilized MutS;

(i) separating unbound DNA from bound DNA contacted in step (h);

(j) releasing bound DNA separated in step (i) from the second immobilized MutS;

(k) contacting DNA released in step (j) with immobilized streptavidin;

(l) denaturing DNA contacted in step (k);

(m) separating bound DNA from unbound DNA denatured in step (l),

which bound DNA separated in step (m) encodes one or more identified affected alleles underlying the disease phenotype.

54. The method of claim 53, wherein releasing bound DNA from the second immobilized MutS in step (j) is carried out using ATP or proteinase K.