Subtractive hybridization techniques for identifying differentially expressed and commonly expressed nucleic acid

• US 5,935,788 A
• Filed: 07/24/1997
• Issued: 08/10/1999
• Est. Priority Date: 06/25/1996
• Status: Expired due to Fees

• Current Assignee: LifeSpan Biosciences Incorporated
• Original Assignee: LifeSpan Biosciences Incorporated
• Inventors: Stewart, Christine C., Burmer, Glenna C., Brown, Joseph P.
• Primary Examiner(s): Ketter, James
• Assistant Examiner(s): SANDALS, WILLIAM O
• Application Number:
  US08/901,224
• Time in Patent Office: 747 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.2, 536/24.33, 536/24.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2537/107 Homoduplex formation
• CPC Class Codes: C12Q 2539/101 Subtraction analysis

Abstract

The present invention provides gene subtraction methods that can be used to identify and isolate from hybridization mixtures differentially expressed nucleic acid and/or commonly expressed nucleic acid between two populations of nucleic acid. The methods of the present invention employ a restriction endonuclease recognition site that is also recognized in whole or in part by a second restriction endonuclease (e.g., HinPI and BssHII or HhaI and HinP1I) to create different ends (e.g., sticky ends) between the first and second nucleic acid populations. In addition, the methods of the present invention use a selective ligation step, a biotinylated nucleotide extension step, or both to differentiate between homoduplexes, which allow for the identification of sequences that are different between the two nucleic acid populations, and heteroduplexes, which allow for the identification of sequences that are common to the two nucleic acid populations.

Claims

1. A method for detecting a target nucleic acid present in a first nucleic acid population (a first nucleic acid sample) and not in a second nucleic acid population (a second nucleic acid sample), said method comprising:

(a) separately fragmenting nucleic acid from said first nucleic acid sample and nucleic acid from said second nucleic acid sample with a sample fragmenting restriction endonuclease to create first nucleic acid sample fragments and second nucleic acid sample fragments, respectively;

(b) ligating a pair of first nucleic acid adaptors onto said first nucleic acid sample fragments, each adaptor having a first restriction endonuclease recognition site that is cleaved by a first restriction endonuclease, and amplifying said first nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of said first nucleic acid adaptors;

(c) ligating a pair of second nucleic acid adaptors onto said second nucleic acid sample fragments, each adaptor having a second restriction endonuclease recognition site which is cleaved by a second restriction endonuclease that recognizes all or a part of said first restriction endonuclease recognition site, but cleaves so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments, and amplifying said second nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of the second nucleic acid adaptors;

(d) fragmenting said first nucleic acid sample fragments with said first restriction endonuclease and said second nucleic acid sample fragments with said second restriction endonuclease;

then

(e) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture; and

(f) isolating a homoduplex from said hybridization mixture, wherein the isolation of said homoduplex identifies target nucleic acid which is present in said first nucleic acid sample and not in said second nucleic acid sample.

2. The method in accordance with claim 1, wherein said nucleic acid is cDNA.

3. The method in accordance with claim 1, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

4. The method in accordance with claim 1, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

5. The method in accordance with claim 1, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are both HinP1I, and said second restriction endonuclease is HhaI.

6. The method in accordance with claim 1, wherein said sample fragmenting restriction endonuclease and said second restriction endonuclease are both HinP1I, and said first restriction endonuclease is HhaI.

7. The method in accordance with claim 1, wherein said sample fragmenting restriction endonuclease is HaeIII, said first restriction endonuclease is KasI, and said second restriction endonuclease is EheI.

8. The method in accordance with claim 1, wherein said homoduplex of step (f) is isolated from said hybridization mixture using a biotinylated nucleotide.

9. The method in accordance with claim 1, wherein said homoduplex of step (f) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.

10. The method in accordance with claim 1, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

11. The method in accordance with claim 10, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

12. A method for detecting a target nucleic acid present in both a first nucleic acid population (a first nucleic acid sample) and a second nucleic acid population (a second nucleic acid sample), said method comprising:

(a) separately fragmenting nucleic acid from said first nucleic acid sample and nucleic acid from said second nucleic acid sample with a sample fragmenting restriction endonuclease to create first nucleic acid sample fragments and second nucleic acid sample fragments, respectively;

(b) ligating a pair of first nucleic acid adaptors onto said first nucleic acid sample fragments, each adaptor having a first restriction endonuclease recognition site that is cleaved by a first restriction endonuclease, and amplifying said first nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of said first nucleic acid adaptors;

(c) ligating a pair of second nucleic acid adaptors onto said second nucleic acid sample fragments, each adaptor having a second restriction endonuclease recognition site which is cleaved by a second restriction endonuclease that recognizes all or a part of said first restriction endonuclease recognition site, but cleaves so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments, and amplifying said second nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of the second nucleic acid adaptors;

(d) fragmenting said first nucleic acid sample fragments with said first restriction endonuclease and said second nucleic acid sample fragments with said second restriction endonuclease;

then

(e) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture; and

(f) isolating a heteroduplex from said hybridization mixture, wherein the isolation of said heteroduplex identifies second target nucleic acid which is present in both said first nucleic acid sample and said second nucleic acid sample.

13. The method in accordance with claim 12, wherein said nucleic acid is cDNA.

14. The method in accordance with claim 12, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

15. The method in accordance with claim 12, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

16. The method in accordance with claim 12, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are both HinpI, and said second restriction endonuclease is HhaI.

17. The method in accordance with claim 12, wherein said sample fragmenting restriction endonuclease and said second restriction endonuclease are both HinP1I, and said first restriction endonuclease is HhaI.

18. The method in accordance with claim 12, wherein said sample fragmenting restriction endonuclease is HaeIII, said first restriction endonuclease is KasI, and said second restriction endonuclease is EheI.

19. The method in accordance with claim 12, wherein said heteroduplex of step (g) is isolated from said hybridization mixture using a biotinylated nucleotide.

20. The method in accordance with claim 12, wherein said heteroduplex of step (g) is isolated from said hybridization mixture by ligating a pair of adaptors onto said heteroduplex.

21. The method in accordance with claim 12, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

22. The method in accordance with claim 21, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

23. A method for detecting a first target nucleic acid present in a first nucleic acid population (a first nucleic acid sample) and not in a second nucleic acid population (a second nucleic acid sample), and a second target nucleic acid present in both said first nucleic acid sample and said second nucleic acid sample, said method comprising:

(a) separately fragmenting nucleic acid from said first nucleic acid sample and nucleic acid from said second nucleic acid sample with a sample fragmenting restriction endonuclease to create first nucleic acid sample fragments and second nucleic acid sample fragments, respectively;

(b) ligating a pair of first nucleic acid adaptors onto said first nucleic acid sample fragments, each adaptor having a first restriction endonuclease recognition site that is cleaved by a first restriction endonuclease, and amplifying said first nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of said first nucleic acid adaptors;

(c) ligating a pair of second nucleic acid adaptors onto said second nucleic acid sample fragments, each adaptor having a second restriction endonuclease recognition site which is cleaved by a second restriction endonuclease that recognizes all or a part of said first restriction endonuclease recognition site, but cleaves so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments, and amplifying said second nucleic acid sample fragments with a primer having a sequence that is complementary to a sequence of the second nucleic acid adaptors;

(d) fragmenting said first nucleic acid sample fragments with said first restriction endonuclease and said second nucleic acid sample fragments with said second restriction endonuclease;

then

(e) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture;

(f) isolating a homoduplex from said hybridization mixture, wherein the isolation of said homoduplex identifies first target nucleic acid present in said first nucleic acid sample and not in said second nucleic acid sample; and

(g) isolating a heteroduplex from said hybridization mixture, wherein the isolation of said heteroduplex identifies second target nucleic acid which is present in both said first nucleic acid sample and said second nucleic acid sample.

24. The method in accordance with claim 23, wherein said nucleic acid is cDNA.

25. The method in accordance with claim 23, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

26. The method in accordance with claim 23, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

27. The method in accordance with claim 23, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are both HinpI, and said second restriction endonuclease is HhaI.

28. The method in accordance with claim 23, wherein said sample fragmenting restriction endonuclease and said second restriction endonuclease are both HinP1I, and said first restriction endonuclease is HhaI.

29. The method in accordance with claim 23, wherein said sample fragmenting restriction endonuclease is HaeIII, said first restriction endonuclease is KasI, and said second restriction endonuclease is EheI.

30. The method in accordance with claim 23, wherein said homoduplex of step (g) is isolated from said hybridization mixture using a biotinylated nucleotide.

31. The method in accordance with claim 23, wherein said homoduplex of step (g) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.

32. The method in accordance with claim 23, wherein said heteroduplex of step (h) is isolated from said hybridization mixture using a biotinylated nucleotide.

33. The method in accordance with claim 23, wherein said heteroduplex of step (h) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.

34. The method in accordance with claim 23, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

35. The method in accordance with claim 34, wherein said sample fragmenting restriction endonuclease and said first restriction endonuclease are the same.

36. A method for detecting a target nucleic acid present in a first nucleic acid population (a first nucleic acid sample) and not in a second nucleic acid population (a second nucleic acid sample), said method comprising:

(a) fragmenting said first nucleic acid sample with a first restriction endonuclease to create first nucleic acid sample fragments and said second nucleic acid sample with a second restriction endonuclease to create second nucleic acid sample fragments, said second restriction endonuclease recognizing all or a part of said first restriction endonuclease recognition site, but cleaving so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments;

then

(b) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture; and

(c) isolating a homoduplex from said hybridization mixture, wherein the isolation of said homoduplex identifies target nucleic acid which is present in said first nucleic acid sample and not in said second nucleic acid sample.

37. The method in accordance with claim 36, wherein said nucleic acid is cDNA.

38. The method in accordance with claim 36, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

39. The method in accordance with claim 36, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

40. The method in accordance with claim 36, wherein said homoduplex of step (c) is isolated from said hybridization mixture using a biotinylated nucleotide.

41. The method in accordance with claim 36, wherein said homoduplex of step (c) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.

42. A method for detecting a target nucleic acid present in both a first nucleic acid population (a first nucleic acid sample) and a second nucleic acid population (a second nucleic acid sample), said method comprising:

(a) fragmenting said first nucleic acid sample with a first restriction endonuclease to creat first nucleic acid sample fragments and said second nucleic acid sample fragments with a second restriction endonuclease to create second nucleic acid sample fragments, said second restriction endonuclease recognizing all or a part of said first restriction endonuclease recognition site, but cleaving so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments;

then

(b) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture; and

(c) isolating a heteroduplex from said hybridization mixture, wherein the isolation of said heteroduplex identifies second target nucleic acid which is present in both said first nucleic acid sample and said second nucleic acid sample.

43. The method in accordance with claim 42, wherein said nucleic acid is cDNA.

44. The method in accordance with claim 42, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

45. The method in accordance with claim 42, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

46. The method in accordance with claim 42, wherein said heteroduplex of step (c) is isolated from said hybridization mixture using a biotinylated nucleotide.

47. The method in accordance with claim 42, wherein said heteroduplex of step (c) is isolated from said hybridization mixture by ligating a pair of adaptors onto said heteroduplex.

48. A method for detecting a first target nucleic acid present in a first nucleic acid population (a first nucleic acid sample) and not in a second nucleic acid population (a second nucleic acid sample), and a second target nucleic acid present in both said first nucleic acid sample and said second nucleic acid sample, said method comprising:

(a) fragmenting said first nucleic acid sample with a first restriction endonuclease to creat first nucleic acid sample fragments and said second nucleic acid sample fragments with a second restriction endonuclease to create second nucleic acid sample fragments, said second restriction endonuclease recognizing all or a part of said first restriction endonuclease recognition site, but cleaving so that different ends are created between said first nucleic acid sample fragments and said second nucleic acid sample fragments;

then

(b) combining said first nucleic acid sample fragments and said second nucleic acid sample fragments under hybridization conditions to form a hybridization mixture;

(c) isolating a homoduplex from said hybridization mixture, wherein the isolation of said homoduplex identifies first target nucleic acid present in said first nucleic acid sample and not in said second nucleic acid sample; and

(d) isolating a heteroduplex from said hybridization mixture, wherein the isolation of said heteroduplex identifies second target nucleic acid which is present in both said first nucleic acid sample and said second nucleic acid sample.

49. The method in accordance with claim 48, wherein said nucleic acid is cDNA.

50. The method in accordance with claim 48, wherein said nucleic acid is derived from a source selected from the group consisting of eukaryotic, prokaryotic, invertebrate, vertebrate, mammalian, non-mammalian and plant.

51. The method in accordance with claim 48, wherein said first restriction endonuclease and said second restriction endonuclease, respectively, are selected from the group consisting of:

HinP1I and HhaI;

HinP1I and CfoI;

Csp6I and RsaI;

HinP1I and BssHII;

MseI and PacI;

AciI and BsrBI;

AciI and SacII;

KasI and EheI;

Acc65I and KpnI;

ApaI and Bsp1201I;

Ppu10I and NsiI; and

SmaI and XmaI.

52. The method in accordance with claim 48, wherein said homoduplex of step (c) is isolated from said hybridization mixture using a biotinylated nucleotide.

53. The method in accordance with claim 48, wherein said homoduplex of step (c) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.

54. The method in accordance with claim 48, wherein said heteroduplex of step (d) is isolated from said hybridization mixture using a biotinylated nucleotide.

55. The method in accordance with claim 48, wherein said heteroduplex of step (d) is isolated from said hybridization mixture by ligating a pair of adaptors onto said homoduplex.