Detection of mismatches by resolvase cleavage using a magnetic bead support

• US 5,851,770 A
• Filed: 10/19/1995
• Issued: 12/22/1998
• Est. Priority Date: 04/25/1994
• Status: Expired due to Fees

• Current Assignee: Variagenics, Inc. (Arca Biopharma, Inc.)
• Original Assignee: Variagenics, Inc. (Arca Biopharma, Inc.)
• Inventors: Babon, Jeff, Stoerker, Jay, Huff, Anne, Youil, Rima, Cotton, Richard G. H.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/545,404
• Time in Patent Office: 1,160 Days
• Field of Search: 435/6, 435/91.2, 435/91.53, 435/810, 435/18, 436/526, 536/24.3, 536/24.33, 536/25.32, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2521/514 Mismatch repair protein
• CPC Class Codes: C12Q 2537/113 Heteroduplex formation
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

Disclosed is a method for detecting one or more mismatches between a first and a second nucleic acid, the first and second nucleic acids being capable of preferentially hybridizing. The method involves: a) providing the first nucleic acid in its single-stranded form, the first nucleic acid being bound to the first member of a specific binding pair; b) providing the second nucleic acid in its single-stranded form, the second nucleic acid being bound to a detectably labelled reagent; c) contacting the first nucleic acid with the second nucleic acid under conditions allowing heteroduplex formation; d) contacting the product of step (c) with a magnetic bead to which is bound the second member of the specific binding pair under conditions allowing complex formation between the first and the second members of the specific binding pair; e) applying a magnetic field to the mixture to facilitate separation of the magnetic bead from the remainder of the product of step (c); f) contacting the magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit the resolvase to cleave the heteroduplex; and f) analyzing the product of step (f), the presence of a cleavage product being an indication of a mismatch between the first and second nucleic acids.

Claims

1. A method for detecting one or more mismatches between a first and a second nucleic acid, said first and said second nucleic acids being capable of preferentially hybridizing, said method comprising:

a) providing a magnetic bead to which is bound, through a specific binding pair, a duplex comprising said first and said second nucleic acids, said duplex being further bound to a detectably labelled reagent;

b) separating said magnetic bead and bound products from the remainder of a sample containing said magnetic beads;

c) contacting said magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit said resolvase to cleave said heteroduplex; and

d) analyzing said product of step (c), the presence of a cleavage product being an indication of a mismatch between said first and said second nucleic acids.

2. The method of claim 1, wherein said magnetic bead is a streptavidin-coated magnetic bead.

3. The method of claim 1, wherein said product of step (f) is analyzed by gel electrophoresis or other detectable signal intensity method.

4. The method of claim 1, wherein said product of step (f) is analyzed by radioactive scintillation counting or fluorescence detection.

5. The method of claim 1, wherein said resolvase is bacteriophage T4 endonuclease VII.

6. The method of claim 1, wherein said first or said second nucleic acid is the product of PCR amplification.

7. The method of claim 6, wherein said first nucleic acid is PCR amplified using at least one primer labelled with said first member of said specific binding pair.

8. The method of claim 6, wherein said first nucleic acid is amplified using one primer labelled with said first member of said specific binding pair and a second primer which is unlabelled.

9. The method of claim 7, wherein said specific binding pair is avidin and biotin.

10. The method of claim 1, wherein said first or said second nucleic acids are rendered single-stranded by denaturation.

11. The method of claim 6, wherein said second nucleic acid is detectably labelled during PCR amplification.

12. The method of claim 11, wherein said detectable label is a radioactive, fluorescent, or other detectable label.

13. The method of claim 1, wherein said mismatch results from a mutation or polymorphism.

14. The method of claim 1, wherein one of said first or said second nucleic acids comprises a reference nucleic acid and the other comprises a test nucleic acid.

27. The method of claims 1, 15, 18, or 19, wherein said first or said second nucleic acid is derived from a eukaryotic cell, a eubacterial cell, a bacterial cell, or a virus.

28. The method of claim 27, wherein said eukaryotic cell is a human cell.

29. The method of claims 1, 15, 18, or 19, or wherein each of said first and said second nucleic acids is at least 100 nucleotides in length.

15. A method for detecting one or more mismatches between a first and a second nucleic acid, said first and said second nucleic acids being capable of preferentially hybridizing, said method comprising:

a) providing a duplex comprising said first and said second nucleic acids, said first nucleic acid being bound to the first member of a specific binding pair;

b) contacting said duplex with a magnetic bead to which is bound a second member of said specific binding pair under conditions allowing complex formation between said first and said second members of said specific binding pair;

c) separating said magnetic bead and bound products from the remainder of said product of step (b);

d) contacting said magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit said resolvase to cleave said heteroduplex; and

e) analyzing the supernatant of step (d), the presence of a cleavage product being an indication of a mismatch between said first and said second nucleic acids.

16. The method of claim 15, wherein said analyzing in step (g) comprises electrophoresis.

17. The method of claim 16, wherein said electrophoresis is followed by silver staining of the electrophoretic gel.

18. A method for detecting one or more mismatches between a first and a second nucleic acid, said first and said second nucleic acids being capable of preferentially hybridizing, said method comprising:

a) providing a duplex comprising said first and said second nucleic acids, said first nucleic acid being bound to the first member of a specific binding pair;

b) contacting said duplex with a magnetic bead to which is bound a second member of said specific binding pair under conditions allowing complex formation between said first and said second members of said specific binding pair;

c) separating said magnetic bead and bound products from the remainder of said product of step (b);

d) contacting said magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit said resolvase to cleave said heteroduplex;

e) labeling the product of step (d) not bound to said magnetic bead with a detectable label; and

f) analyzing said labeled product, the presence of a cleavage product being an indication of a mismatch between said first and said second nucleic acids.

19. A method for detecting one or more mismatches between a first and a second nucleic acid, said first and said second nucleic acids being capable of preferentially hybridizing, said method comprising:

a) providing a magnetic bead to which is bound, through a specific binding pair, a duplex comprising said first and said second nucleic acids, said duplex being further bound to a detectably labelled reagent;

b) separating said magnetic bead and bound products from the remainder of a sample containing said magnetic beads;

c) contacting said magnetic bead-bound nucleic acid with a resolvase capable of recognizing at least one single base pair mismatch in a heteroduplex, under conditions which permit said resolvase to cleave said heteroduplex; and

d) analyzing the product found in the sample supernatant of step (c), the presence of a cleavage product being an indication of a mismatch between said first and said second nucleic acids.

20. A kit for detecting a mismatch in a test nucleic acid, said kit comprising:

a) a magnetic bead to which is bound one member of a specific binding pair; and

b) a resolvase which is capable of recognizing and cleaving at least one single base pair mismatch in a heteroduplex.

21. The kit of claim 20, further comprising a reference nucleic acid which preferentially hybridizes to said test nucleic acid, said reference nucleic acid being capable of binding to the second member of said specific binding pair.

22. The kit of claim 20, further comprising a pair of PCR primers, at least one primer of said pair being capable of binding to said second member of said specific binding pair.

23. The kit of claim 20, further comprising a pair of PCR primers, at least one primer of said pair being bound to a detectable label.

24. The kit of claim 20, wherein said specific binding pair is avidin and biotin.

25. The kit of claim 20, wherein said resolvase is bacteriophage T4 endonuclease VII.

26. The kit of claim 20, wherein said magnetic bead is a streptavidin-coated magnetic bead.