Isothermal nucleic acid amplification methods and compositions

• US 8,034,568 B2
• Filed: 02/12/2009
• Issued: 10/11/2011
• Est. Priority Date: 02/12/2008
• Status: Expired due to Fees

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith, Wang, Shenglong
• Primary Examiner(s): Horlick; Kenneth R.
• Assistant Examiner(s): Tung; Joyce
• Application Number:
  US12/370,534
• Publication Number:
  US 20090203085A1
• Time in Patent Office: 971 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 435/6.12
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid

Abstract

Methods and compositions are provided related to the amplification of target polynucleotide sequences as well as total RNA and total DNA amplification. In some embodiments, the methods and compositions also allow for the immobilization and capture of target polynucleotides with defined 3′ and or 5′ sequences to solid surfaces. The polynucleotides attached to the solid surfaces can be amplified or eluted for downstream processing. In some cases, nucleotides attached to solid surfaces can be used for high throughput sequencing of nucleotide sequences related to target DNA or target RNA.

Claims

1. A method for creating a partial RNA-DNA heteroduplex comprising:

a) providing a template nucleic acid in a reaction mixture;

b) adding a first primer to said reaction mixture and generating a first primer extension product with said first primer;

c) adding a second primer that is an RNA-DNA chimeric primer to said reaction mixture;

d) adding a DNA polymerase to said reaction mixture, wherein said DNA polymerase comprises RNA-dependent polymerase activity; and

e) degrading a portion of said first primer extension product that is not hybridized to a second primer extension product in a double-stranded first and second primer extension product complex by adding an exonuclease to said reaction mixture;

whereby said partial RNA-DNA heteroduplex is created.

2. A method for producing amplified nucleic acid comprising:

a) providing a template nucleic acid;

b) annealing an all-DNA first primer or set of all-DNA first primers comprising a 3′

end whereby a portion of the 3′

end comprises a template annealing sequence and a 5′

tail sequence whereby a portion of the tail sequence comprises a sequence (A);

c) extending said first primer or set of first primers with a DNA polymerase to create a first primer extension product or first set of primer extension products;

d) dissociating the primer extension product or products from the template nucleic acid;

e) annealing a second primer comprising a 3′

DNA sequence and a 5′

RNA sequence, whereby a portion of the 3′

end comprises a randomized annealing sequence and a portion of the 5′

end comprises sequence (A) or a sequence (B), to said primer extension product or products, wherein sequence (B) is different from sequence (A); and

f) extending said second primer with a DNA polymerase to create a second primer extension product or set of second primer extension products to produce a double stranded DNA product or products comprising an A-A′

DNA duplex at one end, wherein (A′

) is the complement of (A).

3. The method of claim 2 wherein the template nucleic acid is selected from the group consisting of DNA and RNA.

4. The method of claim 2 wherein the template nucleic acid is RNA and the DNA polymerase of step (c) is an RNA dependent DNA polymerase.

5. The method of claim 4 wherein the dissociation step of step (d) is performed by degrading the RNA.

6. The method of claim 2 wherein the template nucleic acid is DNA and the DNA polymerase of step (c) is a DNA dependent DNA polymerase.

7. The method of claim 6 wherein the dissociation step of step (d) is performed by heat or chemical denaturation.

8. The method of claim 2 wherein the DNA polymerase does not exhibit substantial strand displacement activity.

9. The method of claim 2 wherein each template annealing sequence of the set of primers is specific for a target or region of template nucleic acid.

10. The method of claim 2 wherein the template annealing sequence of the first primer comprises a random sequence.

11. The method of claim 2 wherein the members of the set of first primers each comprises a distinct 3′

DNA annealing sequence, each specific for a target or region of template nucleic acid.

12. The method of claim 2 wherein the first primer tail sequence is not complementary to the template nucleic acid.

13. The method of claim 2 wherein the 5′

tail sequence of the second primer comprises sequence (A).

14. The method of claim 2 wherein the 5′

tail sequence of the second primer does not comprise tail sequence (A).

15. The method of claim 2, wherein sequence A or B of said second primer is not hybridized to said first primer extension product, and further comprising step (g), degrading single stranded 3′

end of said first primer extension product or products in the complex of first and second primer extension products with a single stranded DNA specific 3′

exonuclease.

16. The method of claim 15 further comprising step (h), adding a DNA polymerase with reverse transcriptase activity to create a double stranded nucleic acid with an A-A′

DNA duplex at one end and an B-B′

DNA-RNA heteroduplex at the other end, wherein sequence B comprises RNA, sequence B′

comprises DNA, and B′

is the complement of B.

17. The method of claim 16 further comprising SPIA with a reaction mixture comprising RNase H, a composite amplification primer comprising a 3′

DNA portion and a 5′

end whereby a portion of the 5′

end comprises RNA wherein the RNase H cleaves the RNA portion of the heteroduplex of step (h) to produce a single stranded end, and the amplification primer hybridizes to the single stranded end produced by RNase H, and a DNA polymerase with strand displacement activity.

18. The method of claim 2 wherein the first primer or set of primers comprises a ligand portion at the 5′

end.

19. The method of claim 16 further comprising:

(i) cleavage of the RNA from the DNA-RNA heteroduplex with RNase H;

(j) annealing a chimeric oligonucleotide comprising a 5′

end whereby the 5′

end comprises RNA sequence (C) and a 3′

end whereby the 3′

end comprises the DNA sequence (B) to the double stranded nucleic acid product;

(k) extending the double stranded nucleic acid product with a DNA polymerase to produce a C-C′

DNA-RNA heteroduplex, wherein C′

is the complement of C; and

(l) amplifying the second primer extension product with a reaction mixture comprising RNase H, an amplification primer comprising a 3′

DNA portion and a 5′

end whereby a portion of the 5′

end comprises RNA that hybridizes to a portion of the single stranded end produced by RNase H, and a DNA polymerase with strand displacement activity to produce an amplification product comprising sequence (B) near its 3′

end and sequence (A′

) at the 5′

end.

32. The method of claim 2 wherein the first primer or set of first primers are immobilized to a solid surface.

33. The method of claim 32 wherein the surface is selected from the group consisting of a bead, a magnetic particle, a microarray, a gene chip, and an array.

34. The method of claim 32 wherein the first primer or set of first primers are attached to the solid surface at the 5′

end.

35. The method of claim 2 wherein the second primer or set of second primers comprise a template annealing sequence.

36. The method of claim 33 wherein the first primer or set of first primers further comprise a spacer element between the surface and the 5′

end.

37. The method of claim 36 wherein the template annealing sequence of the first primer or set of first primers are each specific to a target or region of the template nucleic acid.

38. The method of claim 36 wherein the method further comprises:

(g) cleaving the spacer element with light or chemical cleavage to release the double stranded nucleic acid from said surface; and

(h) amplifying the released double stranded nucleic acid in solution with a reaction mixture comprising RNase H, an amplification primer comprising a DNA portion and a 5′

RNA portion, and a DNA polymerase with strand displacement activity to produce an amplified DNA product or products having a defined 3′

sequence comprising sequence (A′

).

39. The method of claim 2 wherein the first primer or set of first primers are in solution.

20. A method for producing amplified nucleic acid comprising:

a) providing a template nucleic acid;

b) annealing an all-DNA first primer or a set of all-DNA first primers comprising a 3′

end whereby a portion of the 3′

end comprises a template annealing sequence and a 5′

end whereby a portion of the 5′

end comprises a DNA tail sequence (A) that is complementary to a sequence (A′

);

c) extending said first primer or set of first primers with a DNA polymerase to create a primer extension product or set of primer extension products;

d) dissociation of the primer extension product or products from the template nucleic acid;

e) annealing a second primer comprising a 3′

DNA end, whereby a portion of the 3′

end comprises a random template annealing sequence, and a 5′

RNA end, whereby a portion of the 5′

end comprises an RNA tail sequence (A) that is complementary to the sequence (A′

), to said primer extension product or products or to said template nucleic acid;

f) extending said second primer with a DNA polymerase to create a second primer extension product or set of second primer extension products, wherein a portion of the 3′

end of said second primer extension product or products comprises sequence (A′

), wherein (A′

) is the complement of (A); and

g) denaturing and annealing the second primer extension product or set of second primer extension products to form a stem loop structure comprising an A-A′

RNA-DNA heteroduplex.

21. The method of claim 20 further comprising the addition of a 3′

single stranded DNA specific exonuclease and single strand specific RNase to degrade unincorporated primers and template nucleic acid.

22. The method of claim 21 further comprising amplification with a reaction mixture comprising RNase H, an amplification primer comprising sequence (A′

), and a DNA polymerase with strand displacement activity, wherein the amplification primer comprises a 3′

-DNA sequence and a 5′

-RNA sequence.

23. The method of claim 20 wherein said template nucleic acid is selected from the group consisting of RNA and DNA.

24. The method of claim 20 wherein the template nucleic acid is RNA and the DNA polymerase of step (c) is an RNA dependent DNA polymerase.

25. The method of claim 20 wherein the template nucleic acid is DNA and the DNA polymerase of step (c) is a DNA dependent DNA polymerase.

26. The method of claim 24 wherein the dissociation step of step (d) is performed by degradation of the RNA.

27. The method of claim 25 wherein the dissociation step of step (d) is performed by chemical or heat denaturation.

28. The method of claim 20 wherein the DNA polymerase does not exhibit substantial strand displacement activity.

29. The method of claim 20 wherein each DNA annealing sequence in the set of first DNA primers is specific for a target or region of the template nucleic acid.

30. The method of claim 20 wherein a portion of the 5′

tail sequence of the first primer is sequence (A) and a portion of the 5′

tail sequence of the second primer is sequence (A).

31. The method of claim 20 wherein the tail sequence is not complementary to the template nucleic acid.

40. The method of claim 20 wherein the first primer or set of first primers are immobilized to a solid surface.