ISOTHERMAL AMPLIFICATION OF NUCLEIC ACID USING PRIMERS COMPRISING A RANDOMIZED SEQUENCE AND SPECIFIC PRIMERS AND USES THEREOF

• US 20110195457A1
• Filed: 02/09/2010
• Published: 08/11/2011
• Est. Priority Date: 02/09/2010
• Status: Abandoned Application

• Current Assignee: General Electric Company
• Original Assignee: General Electric Company
• Inventors: Zhao, Ming, Gao, Wei, Nelson, John Richard
• Application Number:
  US12/702,884
• Publication Number:
  US 20110195457A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

Methods and kits for amplifying a nucleic acid under isothermal conditions to form an amplified nucleic acid sequence are provided. The methods and kits comprises providing a nucleic acid template, a DNA polymerase, deoxyribonucleoside triphosphates, a primer comprising a randomized sequence, and a specific primer, and amplifying the nucleic acid template.

Claims

1. A method for amplifying a nucleic acid, comprising:

providing a nucleic acid template, a DNA polymerase, deoxyribonucleoside triphosphates, a primer comprising a randomized sequence, and a specific primer; and

amplifying the nucleic acid template under isothermal conditions to form an amplified nucleic acid sequence.

2. The method of claim 1, wherein the random primer comprises at least one modified nucleic acid base.

3. The method of claim 2, wherein the modified nucleic acid base increases the melting temperature of the primer comprising a randomized sequence.

4. The method of claim 2, wherein the modified nucleic acid base is selected from a locked nucleic acid base, a peptide nucleic acid base, or a ribonucleic acid base.

5. The method of claim 1, wherein the specific primer is attached to a first substrate.

6. The method of claim 5, wherein the first substrate is selected from a bead, a test tube, a multi-well plate, or a slide.

7. The method of claim 5, wherein a material of the first substrate is selected from polymer, glass, or metal.

8. The method of claim 5, further comprising capturing the amplified nucleic acid sequence by hybridization of the amplified nucleic acid sequence with the specific primer to form a first substrate-bound nucleic acid sequence.

9. The method of claim 8, further comprising extending a nucleic acid sequence from the hybridization site of the specific primer using the first substrate-bound nucleic acid sequence as a template.

10. The method of claim 8, further comprising amplifying the first substrate-bound nucleic acid sequence by a primer comprising a randomized sequence to form a second amplified nucleic acid sequence.

11. The method of claim 10, further comprising capturing the second amplified nucleic acid sequence by a second substrate by hybridization of the second amplified nucleic acid with a specific primer attached to the second substrate.

12. The method of claim 1, wherein the specific primer is attached to a capturing agent.

13. The method of claim 12, wherein the capturing agent comprises an affinity tag.

14. The method of claim 1, wherein the amplification is performed in an emulsion.

15. The method of claim 1, wherein the amplification comprises a rolling circle amplification or a multiple displacement amplification.

16. The method of claim 1, wherein the amplified nucleic acid sequence is a tandem repeat nucleic acid sequence.

17. The method of claim 1, wherein the nucleic acid template is a circular nucleic acid.

18. The method of claim 1, wherein the nucleic acid template comprises a recombination site.

19. The method of claim 1, wherein the nucleic acid template is a DNA.

20. The method of claim 1, wherein the DNA polymerase is a Phi 29 DNA polymerase.

21. The method of claim 1, wherein the random primer comprises at least 5 nucleotides.

22. A kit for amplifying a nucleic acid comprising:

a Phi29 DNA polymerase;

at least one primer comprising a randomized sequence; and

at least one specific primer.

23. The kit of claim 22, wherein the specific primer is attached to a substrate or a capturing agent.