Cleaved amplified modified polymorphic sequence detection methods

• US 6,110,709 A
• Filed: 12/12/1997
• Issued: 08/29/2000
• Est. Priority Date: 03/18/1994
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Mindrinos, Michael, Ausubel, Frederick M.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/989,883
• Time in Patent Office: 991 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/23.2, 536/25.3, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/125 Electrophoretic separation
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention features methods for detecting polymorphic restriction sites and single nucleotide polymorphisms in nucleic acid molecules and kits for carrying out these methods.

Claims

1. A method for detecting the presence or absence of a single nucleotide polymorphism in a nucleic acid molecule, said method comprising the steps of:

(a) providing a nucleic acid molecule comprising a first strand, a second strand, and a nucleotide position suspected of comprising a polymorphism;

(b) amplifying a portion of said nucleic acid molecule by PCR using a first primer and a second primer, wherein (i) said first primer comprises a 3'"'"' region that is complementary to a first region of said first strand, the 5'"'"' end of said first region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said first primer comprising at least one nucleotide mismatch with said first region within 15 nucleotides of said nucleotide position, (ii) said second primer comprises a 3'"'"' region that is complementary to a second region of said second strand, the 5'"'"' end of said second region being positioned 3'"'"' of said nucleotide position, and (iii) said amplification generates a PCR product comprising at least one point mutation, relative to the sequence of said nucleic acid molecule, that forms a portion of a restriction endonuclease recognition site in the presence of a single polymorphic nucleotide in said nucleotide position;

(c) labeling one end of one strand of the product of step (b) with a detectable label to generate a labeled PCR product comprising a labeled strand;

(d) treating said labeled PCR product with a restriction endonuclease that recognizes said restriction endonuclease recognition site to generate a digestion product;

(e) denaturing and contacting said digestion product with a probe that (i) is complementary to a first segment of said labeled strand that is on the opposite side of said nucleotide position from said detectable label, within said labeled strand, (ii) is not complementary to said labeled strand between said nucleotide position and said detectable label, and (iii) is immobilized to a binding element on a solid support; and

(f) assaying for said detectable label bound to said binding element, wherein the absence of said label bound to said binding element indicates the presence of said single nucleotide polymorphism in said nucleic acid molecule, and the presence of said label bound to said binding element indicates the absence of said single nucleotide polymorphism in said nucleic acid molecule.

2. The method of claim 1, wherein step (b) further comprises amplifying said PCR product by PCR using a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a third region of said first strand, the 5'"'"' end of said third region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said third primer comprising an additional nucleotide mismatch with said third region within 15 nucleotides of said nucleotide position, (ii) said fourth primer comprises a 3'"'"' region that is complementary to a fourth region of said second strand, the 5'"'"' end of said fourth region being positioned 3'"'"' of said nucleotide position, and (iii) said amplification generates a PCR product comprising an additional point mutation, relative to the sequence of said nucleic acid molecule, that forms an additional portion of said restriction endonuclease recognition site in the presence of said single nucleotide polymorphism.

3. The method of claim 1, wherein step (b) further comprises amplifying said PCR product by PCR using a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a third region of said first strand, the 5'"'"' end of said third region being positioned 3'"'"' of said nucleotide position, (ii) said fourth primer comprises a 3'"'"' region that is complementary to a fourth region of said second strand, the 5'"'"' end of said fourth region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said fourth primer comprising an additional nucleotide mismatch with said fourth region within 15 nucleotides of said nucleotide position, and (iii) said amplification generates a PCR product comprising an additional point mutation, relative to the sequence of said nucleic acid molecule, that forms an additional portion of said restriction endonuclease recognition site in the presence of said single nucleotide polymorphism.

4. The method of claim 1, wherein said labeling of said one end of said one strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a region of said first strand and that is on the same side of said nucleotide position as said first region of said first strand, (ii) said fourth primer comprises a 3'"'"' region that is complementary to a region of said second strand and that is on the same side of said nucleotide position as said second region of said second strand, and (iii) either said third primer or said fourth primer comprises a detectable label.

5. The method of claim 1, wherein said first primer further comprises a tag element that is not complementary to said first strand of said nucleic acid molecule, said tag element being positioned 5'"'"' to said 3'"'"' region of said first primer.

6. The method of claim 5, wherein said first primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complementary to said first strand of said nucleic acid molecule and is positioned 5'"'"' to said tag element.

7. The method of claim 6, wherein said labeling of said 5'"'"' end of said first strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a third and a fourth primer, wherein (i) the 3'"'"' region of said third primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said fourth primer comprises a 3'"'"' region that is complementary to a region of said second strand that is 3'"'"' of said nucleotide position.

8. The method of claim 1, wherein said second primer further comprises a tag element that is not complementary to said second strand of said nucleic acid molecule, said tag element being positioned 5'"'"' to said 3'"'"' region of said second primer.

9. The method of claim 8, wherein said second primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complemenary to said second strand of said nucleic acid and is positioned 5'"'"' to said tag element.

10. The method of claim 9, wherein said labeling of said 5'"'"' end of said second strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a third and a fourth, wherein (i) the 3'"'"' region of said third primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said fourth primer comprises a 3'"'"' region that is complementary to a region of said first strand that is 3'"'"' of said nucleotide position.

11. The method of claim 2, wherein said third primer further comprises a tag element that is not complementary to said first strand of said nucleic acid molecule and is positioned 5'"'"' to said 3'"'"' region of said third primer.

12. The method of claim 11, wherein said third primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complementary to said first strand of said nucleic acid molecule and is positioned 5'"'"' to said tag element.

13. The method of claim 12, wherein said labeling of said 5'"'"' end of said first strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a fifth and a sixth primer, wherein (i) the 3'"'"' region of said fifth primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said sixth primer comprises a 3'"'"' region that is complementary to a region of said second strand that is 3'"'"' of said nucleotide position.

14. The method of claim 2, wherein said fourth primer further comprises a tag element that is not complementary to said second strand of said nucleic acid molecule and is positioned 5'"'"' to said 3'"'"' region of said fourth primer.

15. The method of claim 14, wherein said fourth primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complementary to said second strand of said nucleic acid molecule and is positioned 5'"'"' to said tag element.

16. The method of claim 15, wherein said labeling of said 5'"'"' end of said second strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a fifth and a sixth primer, wherein (i) the 3'"'"' region of said fifth primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said sixth primer comprises a 3'"'"' region that is complementay to a region of said first strand that is 3'"'"' of said nucleotide position.

17. The method of claim 3, wherein said third primer further comprises a tag element that is not complementary to said first strand of said nucleic acid molecule and is positioned 5'"'"' to said 3'"'"' region of said third primer.

18. The method of claim 17, wherein said third primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complementary to said first strand of said nucleic acid molecule and is positioned 5'"'"' to said tag element.

19. The method of claim 18, wherein said labeling of said 5'"'"' end of said first strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a fifth and a sixth primer, wherein (i) the 3'"'"' region of said fifth primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said sixth primer comprises a 3'"'"' region that is complementary to a region of said second strand that is 3'"'"' of said nucleotide position.

20. The method of claim 3, wherein said fourth primer further comprises a tag element that is not complementary to said second strand of said nucleic acid molecule and is positioned 5'"'"' to said 3'"'"' region of said fourth primer.

21. The method of claim 20, wherein said fourth primer further comprises a binding site for a universal primer for amplification, wherein said binding site is not complementary to said second strand of said nucleic acid molecule and is positioned 5'"'"' to said tag element.

22. The method of claim 21, wherein said labeling of said 5'"'"' end of said second strand of said product of step (b) is carried out by amplifying by PCR said product of step (b) using a fifth and sixth primer, wherein (i) the 3'"'"' region of said fifth primer comprises a region having the sequence of said binding site for said universal primer, and (ii) said sixth primer comprises a 3'"'"' region that is complementary to a region of said first strand that is 3'"'"' of said nucleotide position.

23. The method of claim 1, 2, or 3, wherein said first primer further comprises a first tag element that is not complementary to said first strand of said nucleic acid molecule, said first tag element being positioned 5'"'"' to said 3'"'"' region of said first primer, and said second primer further comprises a second tag element that is not complementary to said second strand of said nucleic acid molecule, said second tag element being positioned 5'"'"' to said 3'"'"' region of said second primer.

24. The method of claim 23, wherein, upon said amplification, said first tag element creates a third tag element in the complementary strand of said PCR product and said second tag element creates a fourth tag element in the complementary strand of said PCR product.

25. The method of claim 24, wherein said solid support comprises four binding elements to each of which is immobilized a probe that binds one of said first tag element, said second tag element, said third tag element, or said fourth tag element.

26. The method of claim 25, wherein step (f) is carried out by assaying for said detectable label bound to each of said four binding elements.

27. The method of claim 1, 2, or 3, wherein said solid support is a chip.

28. A method for detecting the presence of a first single nucleotide polymorphism or a second single nucleotide polymorphism at a nucleotide position in a nucleic acid molecule, said method comprising the steps of:

(a) providing a first aliquot of a nucleic acid molecule, said nucleic acid molecule comprising a first strand, a second strand, and a nucleotide position suspected of comprising a polymorphism;

(b) amplifying a portion of said nucleic acid molecule by PCR using a first primer and a second primer, wherein (i) said first primer comprises a 3'"'"' region that is complementary to a first region of said first strand, the 5'"'"' end of said first region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said first primer comprising at least one nucleotide mismatch with said first region within 15 nucleotides of said nucleotide position, (ii) said second primer comprises a 3'"'"' region that is complementary to a second region of said second strand, the 5'"'"' end of said second region being positioned 3'"'"' of said nucleotide position, and (iii) said amplification generates a first PCR product comprising at least one point mutation, relative to the sequence of said nucleic acid molecule, that forms a portion of a first restriction endonuclease recognition site in the presence of a first single polymorphic nucleotide in said nucleotide position;

(c) labeling one end of one strand of the product of step (b) with a detectable label to generate a first labeled PCR product comprising a labeled strand;

(d) treating said first labeled PCR product with a first restriction endonuclease that recognizes said first restriction endonuclease recognition site to generate a first digestion product;

(e) denaturing and contacting said first digestion product with a first probe that (i) is complementary to a first segment of said labeled strand that is on the opposite side of said nucleotide position from said detectable label, within said labeled strand, (ii) is not complementary to said labeled strand between said nucleotide position and said detectable label, and (iii) is immobilized on a first binding element;

(f) assaying for said detectable label bound to said first binding element;

(g) providing a second aliquot of said nucleic acid molecule;

(h) amplifying a portion of said nucleic acid molecule by PCR using a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a first region of said first strand, the 5'"'"' end of said first region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said third primer comprising at least one nucleotide mismatch with said first region within 15 nucleotides of said nucleotide position, (ii) said fourth primer comprises a 3'"'"' region that is complementary to a second region of said second strand, the 5'"'"' end of said second region being positioned 3'"'"' of said nucleotide position, and (iii) said amplification generates a second PCR product comprising at least one point mutation, relative to the sequence of said nucleic acid molecule, that forms a portion of a second restriction endonuclease recognition site in the presence of a second single polymorphic nucleotide in said nucleotide position;

(i) labeling one end of one strand of the product of step (h) with a detectable label to generate a second labeled PCR product comprising a labeled strand;

(j) treating said second labeled PCR product with a second restriction endonuclease that recognizes said second restriction endonuclease recognition site to generate a second digestion product;

(k) denaturing and contacting said second digestion product with a second probe that (i) is complementary to a first segment of said labeled strand that is on the opposite side of said nucleotide position from said detectable label, within said labeled strand, (ii) is not complementary to said labeled strand between said nucleotide position and said detectable label, and (iii) is immobilized on a second binding element; and

(l) assaying for said detectable label bound to said second binding element,

wherein (i) the absence of said label bound to said first binding element indicates the presence of said first single nucleotide polymorphism;

(ii) the absence of said label bound to said second binding element indicates the presence of said second single nucleotide polymorphism; and

(iii) the absence of said label bound to both of said first binding element and said second binding element indicates the presence of said first single nucleotide polymorphism and said second single nucleotide polymorphism.

29. A kit for detecting the presence or absence of a single nucleotide polymorphism in a nucleic acid molecule comprising:

(a) a first primer and a second primer flanking a nucleotide position suspected of comprising a polymorphism, wherein (i) said first primer comprises a 3'"'"' region that is complementary to a first region of a first strand of said nucleic acid molecule, the 5'"'"' end of said first region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said first primer comprising at least one nucleotide mismatch with said first region within 15 nucleotides of said nucleotide position, and (ii) said second primer comprises a 3'"'"' region that is complementary to a second region of a second strand of said nucleic acid molecule, the 5'"'"' end of said second region being positioned 3'"'"' of said nucleotide position, wherein amplifying said nucleic acid molecule using said first primer and said second primer generates a PCR product comprising at least one point mutation, relative to the sequence of said nucleic acid molecule, that forms a portion of a restriction endonuclease recognition site in the presence of a single polymorphic nucleotide in said nucleotide position; and

(b) a probe that either (i) is complementary to a first segment of said second strand and that is on the opposite side of said nucleotide position from said first primer and is not complementary to said second strand between said nucleotide position and said first primer, or (ii) is complementary to a first segment of said first strand that is on the opposite side of said nucleotide position from said second primer and is not complementary to said first strand between said nucleotide position and said second primer, said probe being immobilized to a binding element on a solid support.

30. The kit of claim 29, wherein either of said first primer or said second primer is tagged with a detectable label.

31. The kit of claim 30, wherein said kit further comprises means for assaying said detectable label.

32. The kit of claim 29, wherein said kit further comprises a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a third region of said first strand, the 5'"'"' end of said third region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said third primer comprising an additional nucleotide mismatch with said third region within 15 nucleotides of said nucleotide position, and (ii) said fourth primer comprises a 3'"'"' region that is complementary to a fourth region of said second strand, the 5'"'"' end of said fourth region being positioned 3'"'"' of said nucleotide position, wherein amplifying said nucleic acid molecule using said third primer and said fourth primer generates a PCR product comprising an additional point relative to the sequence of said nucleic acid molecule, that form an additional portion of said restriction endonuclease recognition site in the presence of a single polymorphic nucleotide in said nucleotide position.

33. The kit of claim 29, wherein said kit further comprises a third primer and a fourth primer, wherein (i) said third primer comprises a 3'"'"' region that is complementary to a third region of said first strand, the 5'"'"' end of said third region being positioned 3'"'"' of said nucleotide position, and (ii) said fourth primer comprises a 3'"'"' region that is complementary to a fourth region of said second strand, the 5'"'"' end of said fourth region being positioned within 15 nucleotides 3'"'"' of said nucleotide position, and said 3'"'"' region of said fourth primer comprising an additional nucleotide mismatch with said fourth region within 15 nucleotides of said nucleotide position, wherein amplifying said nucleic acid molecule using said third primer and said fourth primer generates a PCR product comprising an additional point mutation, relative to the sequence of said nucleic acid molecule, that forms an additional portion of said restriction endonuclease recognition site in the presence of a single polymorphic nucleotide in said nucleotide position.

34. The kit of claim 32 or 33, wherein either of said third primer or said fourth primer is tagged with a detectable label.

35. The kit of claim 34, wherein said kit further comprises means for assaying said detectable label bound to said binding element.