In vitro amplification of nucleic acid molecules via circular replicons

• US 6,448,017 B1
• Filed: 07/09/2001
• Issued: 09/10/2002
• Est. Priority Date: 08/04/1992
• Status: Expired due to Fees

• Current Assignee: Replicon Incorporated (Roper Technologies, Inc.)
• Original Assignee: Replicon Incorporated (Roper Technologies, Inc.)
• Inventors: Auerbach, Jeffrey I.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/899,834
• Publication Number:
  US 20020102575A1
• Time in Patent Office: 428 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 536/23.1, 536/24.3
• US Class Current: 435/6.18
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2527/101 Temperature
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation

Abstract

Methods and compositions suitable for accomplishing the in vitro amplification of nucleic acid molecules via enzymatic means are provided. The preferred means employ circular rather than linear replicons. Means for producing such circular replicons from linear reactants are also provided.

Claims

1. An in vitro composition for exponentially amplifying in vitro a target polynucleotide region of a nucleic acid molecule, wherein said composition comprises:

(A) a single-stranded first polynucleotide, wherein said polynucleotide (i) contains a polynucleotide region that is complementary in sequence to said target polynucleotide region, and (ii) is a circular polynucleotide or is circularizable when hybridized to a polynucleotide comprising said target polynucleotide region;

(B) a second polynucleotide said second polynucleotide being a primer having a 3′

terminus hybridized to said single-stranded first polynucleotide and comprising said target polynucleotide region;

(C) a third polynucleotide, said third polynucleotide being a primer and comprising a polynucleotide region that is complementary in sequence to a region of said second polynucleotide, and whose 3′

terminus is hybridized thereto; and

(D) a polymerase, wherein said polymerase extends the 3′

termini of said second and third polynucleotides in a template-dependent manner to thereby provide said exponential amplification of said target polynucleotide region.

2. The composition of claim 1, wherein said single-stranded first polynucleotide is a circular polynucleotide.

3. The composition of claim 1, wherein said single-stranded first polynucleotide is circularizable when hybridized to said target polynucleotide region.

4. The composition of claim 3, wherein said single-stranded first polynucleotide is circularizable via the action of a ligase.

5. The composition of claim 3, wherein said single-stranded first polynucleotide is circularizable via the action of a recombinase.

6. The composition of claim 1, wherein said polymerase is additionally capable of causing extension-dependent strand displacement of hybridized polynucleotides.

7. The composition of claim 6, wherein said single-stranded first polynucleotide is circular.

8. The composition of claim 6, wherein said single-stranded first polynucleotide is circularizable.

9. The composition of claim 8, wherein said single-stranded first polynucleotide is circularizable via the action of a ligase.

10. The composition of claim 8, wherein said single-stranded first polynucleotide is circularizable via the action of a recombinase.

11. The composition of claim 1, wherein said single-stranded first polynucleotide contains a modified nucleotide.

12. The composition of claim 11, wherein said modified nucleotide is a ribonucleotide.

13. The composition of claim 11, wherein said modified nucleotide is a biotinylated nucleotide.

14. A kit for exponentially amplifying, in vitro, a target polynucleotide region of a nucleic acid molecule, wherein said kit comprises:

(A) a first container, said first container containing a single-stranded first polynucleotide, wherein said polynucleotide (i) contains a polynucleotide region that is complementary in sequence to said target polynucleotide region, and (ii) is a circular polynucleotide or is circularizable when hybridized to a polynucleotide comprising said target polynucleotide region; and

(B) a second container, said second container containing a second polynucleotide, said second polynucleotide being a primer having a 3′

terminus hybridized to said single-stranded first polynucleotide and comprising said target polynucleotide region; and

(C) a third container, said third container containing a third polynucleotide, said third polynucleotide being a primer and comprising a polynucleotide region that is complementary in sequence to a region of said second polynucleotide, and whose 3′

terminus is hybridized thereto;

wherein, in the presence of a polymerase, said 3′

termini of said second and third polynucleotides are extended in a template-dependent manner to hereby provide said exponential amplification of said target polynucleotide region.

15. The kit of claim 14, wherein said single-stranded first polynucleotide is a circular polynucleotide.

16. The kit of claim 14, wherein said single-stranded first polynucleotide is circularizable when hybridized to said target polynucleotide region.

17. The kit of claim 16, wherein said single-stranded first polynucleotide is circularizable via the action of a ligase.

18. The kit of claim 16, wherein said single-stranded first polynucleotide is circularizable via the action of a recombinase.

19. The kit of claim 14, wherein said reagents additionally comprise a fourth container, said fourth container containing a template-dependent polymerase sufficient to extend a 3′

terminus of a polynucleotide hybridized to said single-stranded first polynucleotide in vitro to thereby produce a template-dependent extension product and wherein said polymerase is additionally capable of causing extension-dependent strand displacement of hybridized polynucleotides.

20. The kit of claim 19 wherein said first or second polynucleotides contain a modified nucleotide.