Cascade nucleic acid amplification reaction

• US 6,610,481 B2
• Filed: 12/17/1999
• Issued: 08/26/2003
• Est. Priority Date: 12/05/1995
• Status: Expired due to Term

• Current Assignee: Jørn Erland Koch
• Original Assignee: Jørn Erland Koch
• Inventors: Koch, Jørn Erland
• Primary Examiner(s): Siew, Jeffrey
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/465,589
• Publication Number:
  US 20020031764A1
• Time in Patent Office: 1,348 Days
• Field of Search: 435/91.2, 435/91.51, 435/91.52, 536/22.1
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

A process for generating multiple linear complements of a single strand, circular nucleic acid template containing at least one cleavage site is described. The process consists of combining the single strand, circular nucleic acid template with polynucleotide primers under conditions sufficient for hybridization; extending the polynucleotide primer more than once around the circle to generate a complementary displacement of more than one continguous complement of the single strand, circular nucleic acid template. Also described is a process of synthesizing novel single strand, circular nucleic acids between 30 an 2200 nucleotides. The process consist of synthesizing a linear polynucleotide; combining the linear polynucleotide with a complementary linking oligonucleotide under conditions sufficient for hybridization; and ligating the linear polynucleotide pto produce a single strand, circular nucleic acid.

Claims

1. A method for amplifying a linear nucleic acid, which process comprises:

(a) hybridizing ends of the linear nucleic acid to a chromosomal locus so that the linear nucleic acid is circularized; and

(b) contacting the circularized nucleic acid with a polymerase capable of strand displacement and with nucleoside triphosphates, so that a 3′

-end of the chromosomal locus serves as a primer for rolling circle replication of the circularized nucleic acid, and wherein copies of the linear nucleic acid are produced by said rolling circle replication.

2. A method according to claim 1 in which the chromosomal locus is detected by detecting the amplified nucleic acid.

3. A method according to claim 1 in which the nucleoside triphosphates are labeled.

4. A method according to claim 3 in which the label is selected from the group consisting of:

an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate, and a hapten.

5. A method according to claim 1 in which the polymerase is without 5′

-3′

exonuclease activity.

6. A method according to claim 1 wherein:

(i) the product of the rolling circle replication contains multiple tandem repeat copies of the linear nucleic acid sequence; and

(ii) said product is further amplified in a cascade reaction that comprises contacting the product with the polymerase in the presence of nucleoside triphosphates and a primer, so that nucleic acid synthesis is initiated at the tandem repeats.

7. A method according to claim 6 in which the linear nucleic acid comprises a sequence having dyad symmetry.

8. A method according to claim 7 in which the sequence having dyad symmetry is the primer for nucleic acid synthesis in the cascade reaction.

9. A method according to claim 6 in which the nucleoside triphosphates are labeled.

10. A method according to claim 9 in which the label is selected from the group consisting of:

an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate, and a hapten.

11. A method according to claim 6 which the polymerase is without 5′

-3′

exonuclease activity.

12. A method for amplifying a linear nucleic acid, which process comprises:

(a)hybridizing ends of the linear nucleic acid to a nucleic acid template so that the linear nucleic acid is circularized, said nucleic acid template being fixed to a solid support; and

(b) contacting the circularized nucleic acid with a polymer capable of strand displacement and with nucleoside triphosphates, so that a 3′

-end of the nucleic acid template serves as a primer for rolling circle replication of the circularized nucleic acid, and wherein the linear nucleic acid is amplified by said rolling circle replication.

13. A method according to claim 12 in which the nucleic acid template is detected by detecting the amplified nucleic acid.

14. A method according to claim 12 in which the nucleoside triphosphates are labeled.

15. A method according to claim 14 in which the label is selected from the group consisting of:

an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate, and a hapten.

16. A method according to claim 12 in which the polymerase is without 5′

-3′

exonuclease activity.

17. A method according to claim 12 wherein:

(i) the product of the rolling circle replication contains multiple tandem repeat copies of the linear nucleic acid sequence; and

(ii) said product is further amplified in a cascade reaction that comprises contacting the product with the polymerase in the presence of nucleoside triphosphates and a primer, so that nucleic acid synthesis is initiated at the tandem repeats.

18. A method according to claim 17 in which the linear nucleic acid comprises a sequence having dyad symmetry.

19. A method according to claim 18 in which the sequence having dyad symmetry is the primer for nucleic acid synthesis in the cascade reaction.

20. A method according to claim 17 in which the nucleoside triphosphates are labeled.

21. A method according to claim 20 in which the label is selected from the group consisting of:

an enzyme, a radioactive isotope, a fluorescent compound, a chemiluminescent compound, a bioluminescent compound, a metal chelate, and a hapten.

22. A method according to claim 17 in which the polymerase is without 5′

-3′

exonuclease activity.