In vitro amplification of nucleic acid molecules via circular replicons

• US 7,615,625 B2
• Filed: 06/29/2007
• Issued: 11/10/2009
• Est. Priority Date: 08/04/1992
• Status: Expired due to Fees

• Current Assignee: Replicon Incorporated (Replicon Software, Inc.)
• Original Assignee: Replicon Incorporated (Replicon Software, Inc.)
• Inventors: Auerbach, Jeffrey I.
• Primary Examiner(s): Horlick; Kenneth R.
• Application Number:
  US11/824,035
• Publication Number:
  US 20080305535A1
• Time in Patent Office: 865 Days
• Field of Search: 435/6, 435/91.2, 536/24.3
• US Class Current: 536/24.3
• 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 2521/507 Recombinase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle

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. A kit for exponentially amplifying, in vitro, a target polynucleotide region of a nucleic acid molecule, wherein the kit comprises one or more containers comprising:

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

(B) a second polynucleotide, wherein the second polynucleotide is a primer having a 3′

terminus hybridized to the single-stranded first polynucleotide and comprises the target polynucleotide region; and

(C) a third polynucleotide, wherein the third polynucleotide is a primer and comprises a polynucleotide region that is complementary in sequence to a region of the second polynucleotide,

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

termini of the second and third polynucleotides are extended in a template-dependent manner to exponentially amplify the target polynucleotide region.

2. The kit of claim 1, wherein the first or second polynucleotide contains a modified nucleotide.

3. The kit of claim 1, wherein the kit further comprises a circularizing agent to form the circular polynucleotide.

4. The kit of claim 3, wherein the circularizing agent is a ligase.

5. The kit of claim 3, wherein the circularizing agent is a recombinase.

6. The kit of claim 1, wherein the polymerase is a strand-displacing polymerase.

7. The kit of claim 1, wherein the kit further comprises a strand-displacing agent.

8. The kit of claim 1, wherein the kit further comprises a restriction endonuclease.

9. The kit of claim 1, wherein the kit further comprises a mixture comprising nucleotides.

10. The kit of claim 9, wherein the mixture comprises ribonucleotides.

11. The kit of claim 9, wherein the mixture comprises a nucleotide analogue.

12. The kit of claim 1, wherein the one or more containers further comprises a template-dependent polymerase sufficient to extend a 3′

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

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

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

15. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a mammal.

16. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a mammalian pathogen.

17. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a virus.

18. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a bacterium.

19. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a fungus.

20. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a procaryotic or eucaryotic pathogen.

21. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule of a parasite.

22. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule that has been chemically synthesized.

23. The kit of claim 1, wherein said single-stranded first polynucleotide comprises a nucleic acid sequence complementary to a nucleic acid molecule from a source selected from the group consisting of procaryotic organisms, pathogenic bacteria, non-pathogenic bacteria, Escherichia, Salmonella, Clostridium, Agrobacter, Staphylococcus, Streptomyces, Streptococcus, Rickettsiae, Chlamydia, Mycoplasma, eucaryotic organisms, protozoans, amoebas, parasites, Plasmodium, Trypanosomes, fungi, yeast, higher plants, lower animals, higher animals, mammals, humans, viruses, Herpes viruses, Human Immunodeficiency Virus (HIV), influenza virus, Epstein-Barr virus, hepatitis virus, polio virus, viroids, and chemically synthesized nucleic acids.