Methods of preparing nucleic acids for mass spectrometric analysis

• US 6,566,055 B1
• Filed: 06/03/1998
• Issued: 05/20/2003
• Est. Priority Date: 09/19/1996
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Shaler, Thomas A., Becker, Christopher H., Tan, Yuping, Monforte, Joseph A.
• Primary Examiner(s): BORIN, MICHAEL L
• Application Number:
  US09/089,730
• Time in Patent Office: 1,812 Days
• Field of Search: 435/86, 435/91.2, 435/6, 250/281
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• 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 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

Abstract

This invention relates to methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods of detecting polymorphisms.

Claims

1. A method of preparing one or more target nucleic acids for mass spectrometric analysis comprising:

(a) identifying one or more target nucleic acids, wherein each of said target nucleic acids comprises a region of interest and two flanking regions,

(b) amplifying at least one target nucleic acid to produce at least one amplified target nucleic acid;

(c) reducing length of at least one of said target nucleic acids by cleaving off at least a portion of both flanking regions to produce one or more reduced-length target nucleic acids, and

(d) determining the masses of each of said reduced-length target nucleic acids using a mass spectrometer.

2. The method of claim 1, wherein at least one of the reduced-length target nucleic acids is single-stranded.

3. The method of claim 1, wherein at least one of the reduced-length target nucleic acids is double-stranded.

4. The method of claim 1, wherein said reducing step further comprises:

cleaving with a restriction endonuclease capable of cleaving at a cleavable site within both flanking regions.

5. The method of claim 4, wherein said restriction endonuclease is a Type IIS restriction endonuclease.

6. The method of claim 4, wherein said restriction endonuclease is a Type II restriction endonuclease.

7. The method of claim 4, wherein one or more recognition sites for said restriction endonuclease is introduced into the two flanking regions by using one or more cleavable primers each comprising said recognition sites during said amplifying step.

8. The method of claim 1, wherein said reducing step comprises treating said amplified target nucleic acid with a 5′

to 3′

exonuclease, wherein a cleavable primer comprising an exonuclease blocking moiety has been introduced during said amplifying step.

9. The method of claim 1, wherein said reducing step further comprises:

cleaving with a chemical capable of cleaving at a cleavable site within both flanking regions.

10. The method of claim 1, further comprising:

purifying one or more reduced-length amplified target nucleic acids.

11. The method of claim 10, wherein the one or more reduced length amplified target nucleic acid comprises a strand of interest hybridized to a complementary strand, and wherein said purifying comprises:

binding the complementary strand of a strand of interest of each of said amplified target nucleic acids to a solid support to produce a bound complementary strand, and

removing at least one strand of interest from said bound complementary strand, wherein the mass of the strand of interest is determined using a mass spectrometer.

12. The method of claim 10, wherein the one or more reduced length amplified target nucleic acid comprises a strand of interest hybridized to a complementary strand, and wherein said purifying comprises:

binding one or more strand of interest to a solid support to produce a bound strand of interest,

removing one or more unbound components comprising said complementary strand by washing, and

releasing said bound strand of interest from said solid support, wherein the mass of the strand of interest is determined using a mass spectrometer.

13. The method of claim 12, wherein said releasing comprises cleaving a cleavable linker.

14. The method of claim 12, wherein said reducing and purifying are the same steps, comprising:

binding one or more of said amplified target nucleic acids to a solid support via a cleavable primer to produce at least one bound amplified target nucleic acid, wherein said cleavable primer has been incorporated during said amplifying step, and

releasing reduced-length amplified target nucleic acids comprising cleaving at one or more cleavable sites within or near said cleavable primer.

15. The method of claim 14, wherein said cleavable primer comprises an exonuclease blocking moiety and wherein said cleaving comprises degrading said bound amplified target nucleic acids with a 5′

-3′

exonuclease, resulting in reduced-length amplified target nucleic acids that are single-stranded.

16. The method of claim 14, wherein said cleavable primer comprises a recognition site for a restriction endonuclease and wherein said cleaving comprises treating said bound amplified target nucleic acid with said restriction endonuclease.

17. The method of claim 10, wherein said purifying comprises:

binding a strand of interest of at least one of said amplified target nucleic acids to a solid support to produce a bound strand of interest and an unbound complementary strand,

removing the unbound strand of each of said bound strands of interest from said bound strand of interest by denaturing and washing; and

releasing said bound strand of interest of said amplified target nucleic acids from said solid support to form single-stranded amplified target nucleic acids, wherein the mass of the single-stranded amplified target nucleic acids is determined using a mass spectrometer.

18. The method of claim 17, wherein said bound strands of said amplified target nucleic acids each comprises a cleavable site within or adjacent to a flanking region of said amplified target nucleic acids.

19. The method of claim 18, wherein said releasing further comprises cleaving said cleavable sites.

20. The method of claim 19, wherein said cleavable sites are introduced into at least one of said amplified target nucleic acids by using a cleavable primer during said amplifying step.

21. The method of claim 19, wherein said cleaving comprises:

cleaving with a restriction endonuclease at a cleavable site within both flanking regions.

22. The method of claim 21, wherein said restriction endonuclease is a Type IIS restriction endonuclease.

23. The method of claim 21, wherein said restriction endonuclease is a Type II restriction endonuclease.

24. The method of claim 21, wherein one or more recognition sites for said restriction endonuclease are introduced into both flanking regions by using one or more cleavable primers each comprising said recognition sites during said amplifying step.

25. The method of claim 19, wherein said cleaving comprises treating said amplified target nucleic acid with a 5′

to 3′

exonuclease, wherein a cleavable primer comprising an exonuclease blocking moiety has been introduced during said amplifying step.

26. The method of claim 19, wherein said cleaving comprises cleaving with a chemical capable of cleaving at a cleavable site within both flanking regions.

27. The method of claim 26, wherein the cleavable site comprises a phosphorothioate linkage.

28. The method of claim 9, wherein the cleavable sites comprises a phosphorothioate linkage.

29. The method of claim 14, wherein the cleavable sites comprises a phosphorothioate linkage.

30. The method of claim 1, wherein at least one of said reduced-length amplified target nucleic acids has one or more nucleotides replaced with mass-modified nucleotides.

31. The method of claim 1, wherein said determining step further comprises utilizing internal self-calibrants to provide improved mass accuracy.

32. A method of determining the mass of one or more target nucleic acids comprising:

(a) amplifying at least one of said target nucleic acids to reproduce an amplified target nucleic acid, wherein said amplified target nucleic acid comprises a strand of interest and a complementary strand, the strand of interest comprising a region of interest and two flanking regions, wherein the complementary strand is hybridized to the strand of interest,

(b) binding the strand of interest to a solid support to produce a bound strand of interest,

(c) removing the complementary strand from the bound strand of interest by denaturing and washing,

(d) releasing the bound strand of interest from the solid support to form a single-stranded amplified target nucleic acid,

(e) reducing the length of the single-stranded amplified target nucleic acid by cleaving off at least a portion of both flanking regions, and

(f) determining-the mass of said reduced-length single-stranded amplified target nucleic acid using a mass spectrometer.

33. The method of claim 32, wherein said reducing step further comprises:

cleaving with a restriction endonuclease capable of cleaving at a cleavable site within or adjacent to both flanking regions of said amplified target nucleic acids.

34. The method of claim 32, wherein said releasing step and said reducing step comprises cleaving said cleavable sites.

35. The method of claim 33, wherein said cleavable sites are introduced into said amplified target nucleic acids by using cleavable primers during said amplifying step.

36. The method of claim 34, wherein said cleaving comprises:

cleaving with a restriction endonuclease at a cleavable site within both flanking regions.

37. The method of claim 36, wherein said restriction endonuclease is a Type IIS restriction endonuclease.

38. The method of claim 36, wherein said restriction endonuclease is a Type II restriction endonuclease.

39. The method of claim 36, wherein one or more recognition sites for said restriction endonuclease is introduced into both flanking regions by using one or more cleavable primers each comprising said recognition sites during said amplifying step.

40. The method of claim 34, wherein said cleaving comprises treating said amplified target nucleic acid with a 5′

to 3′

exonuclease, wherein a cleavable primer comprising an exonuclease blocking moiety has been introduced during said amplifying step.

41. The method of claim 34, wherein said cleaving comprises cleaving with a chemical capable of cleaving at a cleavable site within both flanking regions.

42. The method of claim 41, wherein the cleavable site comprises a phosphorothioate linkage.

43. The method of claim 32, wherein said releasing comprises using a cleavable linker.

44. The method of claim 32, wherein said determining does not involve sequencing of said amplified target nucleic acids.

45. A method of determining the mass of a target nucleic acid which includes a region of interest and two flanking regions, which comprises:

(a) reducing the length of the target nucleic acid by removing at least a portion of both of the flanking regions;

(b) isolating the reduced-length target nucleic acid from its complementary strand; and

(c) determining the mass of the isolated, reduced-length target nucleic acid by mass spectrometry.

46. The method of claim 45, wherein the target nucleic acid is single-stranded.

47. The method of claim 45, wherein the target nucleic acid is double-stranded and the isolated, reduced-length target nucleic acid comprises one of the two strands.

48. A method of determining the mass of one or more target nucleic acids comprising:

(a) identifying one or more target nucleic acids, wherein each of said target nucleic acids comprises a region of interest, at least one Type IIs restriction site, and at least two flanking regions;

(b) amplifying at least on target nucleic acid to produce at least one amplified target nucleic acid;

(c) reducing the length of at least one of said amplified target nucleic acids by cleaving off at least a portion of both flanking regions with a Type IIs restriction endonuclease to produce one or more reduced-length target nucleic acids; and

(d) determining the mass of at least one of said reduced-length, target nucleic acids using a mass spectrometer.