Nucleic acid amplification

• US 20030099937A1
• Filed: 08/15/2002
• Published: 05/29/2003
• Est. Priority Date: 08/15/2001
• Status: Abandoned Application

• Current Assignee: Linden Technologies Incorporated
• Original Assignee: Linden Technologies Incorporated
• Inventors: Law, Simon W.
• Application Number:
  US10/219,616
• Publication Number:
  US 20030099937A1
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6865 Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

Disclosed is a method of amplifying nucleic acids by appending a promoter sequence on an oligonucleotide and transcribing the nucleic acid. The oligonucleotide can attached to a solid phase, e.g., a chip. In one example, nucleic acids are amplified by a method that includes: providing a first solid support having 5′ attached oligonucleotide; annealing a complex sample that comprises sample nucleic acids to the solid support; and producing template nucleic acids immobilized on the solid support that each include at least a segment of the sample nucleic acids, such that the immobilized templates represent the composition of the sample nucleic acids.

Claims

1. A method of producing RNA replicates, the method comprising:

providing a first solid support having attached oligonucleotides that comprise a promoter sequence and a target binding sequence;

annealing a sample that comprises RNAs to the solid support;

extending the attached oligonucleotides using an RNA-directed DNA polymerase to construct DNA replicates of the RNAs;

synthesizing DNA strands complementary to the DNA replicates, thereby producing a double-stranded template that includes the promoter sequence;

joining an adaptor that comprises a tag sequence to the double-stranded template, and transcribing the double-stranded template using an RNA polymerase that recognizes the promoter region to produce first-stranded RNA replicates.

2. The method of claim 1 wherein the double-stranded templates incorporate the attached oligonucleotides and are immobilized to the solid support by the attached oligonucleotides.

3. The method of claim 1 further comprising annealing the first-stranded RNA replicates to immobilized second oligonucleotides that comprise a second promoter sequence and a sequence complementary to the tag sequence.

4. The method of claim 3 further comprising

extending the immobilized second oligonucleotides using an RNA-directed DNA polymerase to construct DNA replicates of the RNAs;

synthesizing DNA strands complementary to the DNA replicates, thereby producing second templates that include the second promoter sequence; and

transcribing the complementary strand using an RNA polymerase that recognizes the second double-stranded promoter region to produce second-stranded RNA replicates.

5. The method of claim 4 wherein the immobilized second oligonucleotides are immobilized to a second solid support.

6. The method of claim 4 wherein the immobilized second oligonucleotides are immobilized to the first solid support.

7. The method of claim 4 further comprising recovering a pool of double-stranded RNA molecules formed from hybridization of the first-stranded and second-stranded RNA replicates.

8. The method of claim 1 wherein the first solid support is a pin.

9. The method of claim 1 wherein the first solid support is an array.

10. The method of claim 1 wherein the first solid support is a surface of a multi-sample carrier.

11. The method of claim 1 wherein the first solid support is a membrane.

12. The method of claim 11 wherein the membrane is disposed in a spin-cup.

13. The method of claim 8 wherein the pin is attached to a base that includes other attached pins, wherein the pins of the base are configured so that they can be disposed into separate reaction mixtures.

14. The method of claim 8 or 13, wherein the pin is transferred between containers during the method, each container comprising different reagents for reaction.

15. The method of claim 4 wherein the second-stranded RNA replicates are used to produce additional first-stranded RNA replicates.

16. A method of providing RNA replicates, the method comprising:

cleaving sample nucleic acids to yield cleaved nucleic acids;

treating the cleaved nucleic acids using a nuclease that preferentially digests double stranded nucleic acid relative to single stranded nucleic acid to yield treated sample nucleic acids;

annealing an oligonucleotide to the treated sample nucleic acids, the oligonucleotide having a promoter region and a target binding region that is complementary to a first target site; and

transcribing the annealed treated sample nucleic acid using an RNA polymerase that recognizes the promoter region to generate RNA replicates.

23. The method of claim 16, 18 or 22 in which the method is substantially isothermal or at temperatures less than about 40°

C.

24. The method of claim 16 or 19 in which the sample nucleic acid comprises genomic DNA.

25. The method of claim 16 or 19 in which the sample nucleic acid comprises cDNA.

28. The method of claim 25 further comprising translating RNA from the transcribing.

30. The method of claim 16 or 29 further comprising generating a DNA copy of an RNA from the transcribing;

and cloning the DNA copy in a vector nucleic acid.

17. A method of producing replicate nucleic acids, the method comprising:

providing a solid support having a plurality of addresses;

at each of the plurality of addresses, depositing or synthesizing an oligonucleotide that includes a 5′

promoter region and a 3′

target binding region that is complementary to a target site;

contacting a sample nucleic acid to the solid support;

for each of the oligonucleotides of the plurality of addresses, permitting the target binding region to anneal to its target site in the sample, if present;

extending the annealed sample nucleic acid using a DNA polymerase; and

transcribing the annealed sample nucleic acid using an RNA polymerase that recognizes the promoter region to produce replicate nucleic acids.

26. The method of claim 17 in which the support is glass or plastic.

27. The method of claim 17, 18, 19 or 21 further comprising storing the support for at least 12 hours after the transcribing;

and repeating the transcribing.

18. A method of producing replicate nucleic acids, the method comprising:

providing a solid support having a plurality of addresses, each address including (1) a first nucleic acid segment having (a) a 5′

promoter region and (b) a variable 3′

target binding region, and (2) a second nucleic acid segment that binds the 5′

promoter region;

annealing sample nucleic acids to the solid support;

joining the 5′

terminus of the second nucleic acid segment to the 3′

end of the annealed sample nucleic acid;

removing unjoined and/or unannealed sample nucleic acids; and

transcribing the joined sample nucleic acids using an RNA polymerase that recognizes the 5′

promoter region to produce replicate nucleic acids.

19. A method of producing replicate nucleic acids, the method comprising:

providing a solid support having a plurality of addresses, each address including a first nucleic acid segment having (a) a 5′

promoter region and (b) a variable 3′

target binding region;

annealing sample nucleic acids to the solid support;

annealing a second nucleic acid segment that binds the 5′

promoter region;

joining the 5′

terminus of the second nucleic acid segment to the 3′

end of an annealed sample nucleic acid;

optionally removing unjoined and/or unannealed sample nucleic acids; and

transcribing the joined sample nucleic acids using an RNA polymerase that recognizes the 5′

promoter region to produce replicate nucleic acids.

20. A method of analyzing genetic polymorphisms comprising:

for each polymorphism, locating a fragment flanked by restriction enzyme sites and including the polymorphism such that the sites are less than about 2000, 1000, 700, 500 nucleotides apart;

synthesizing a promoter oligonucleotide having (a) a 5′

promoter region and (b) a variable 3′

target binding region, the variable 3′

target binding region being near or flanking one of fragment termini;

optionally attaching the promoter oligonucleotide to a solid support;

annealing sample nucleic acid to the promoter oligonucleotides;

contacting a DNA polymerase to the annealed sample nucleic acids to extend the annealed sample nucleic acid and render the promoter double-stranded; and

transcribing the extended annealed sample nucleic acid using an RNA polymerase specific for the promoter.

21. A method of analyzing genetic polymorphisms comprising:

for each polymorphism, synthesizing a promoter oligonucleotide on a solid support, the promoter oligonucleotide having (a) a 5′

terminus attached to the support;

(b) a 5′

promoter region and (c) a variable 3′

target binding region, the variable 3′

target binding region being within 1000 nucleotides (e.g., less than 800, 700, 500, or 400 nucleotides) of the polymorphism;

annealing sample nucleic acid to the promoter oligonucleotides;

contacting a DNA polymerase to the annealed sample nucleic acids to extend the annealed sample nucleic acid and render the promoter double-stranded; and

transcribing the extended annealed sample nucleic acid using an RNA polymerase specific for the promoter.

22. A method comprising:

annealing a nucleic acid strand to a first oligonucleotide that binds to the target strand;

extending the target strand 3′

end to form a first oligonucleotide-strand complex;

transcribing the first oligonucleotide-strand complex using a first RNA polymerase to yield a first RNA strand;

annealing the first RNA strand to a second oligonucleotide that binds to the first RNA strand;

reverse transcribing the first RNA strand to yield to a first copy strand;

rendering the first copy strand double-stranded to form a second oligonucleotide-copy strand complex; and

transcribing the second oligonucleotide-copy strand complex, wherein the first oligonucleotide includes a promoter region, specifically recognized by a first RNA polymerase, and a target binding region that binds the target strand 3′

end, and the second oligonucleotide includes a promoter region, specifically recognized by a second RNA polymerase, and a target binding region that binds the first RNA strand 3′

end.

29. The method of claim 22 further comprising joining an adaptor sequence to the first oligonucleotide-strand complex prior to the transcribing.

31. A method of producing RNA replicates, the method comprising:

providing a solid support having attached oligonucleotides;

annealing a sample that comprises RNAs to the solid support;

extending the attached oligonucleotides using an RNA-directed DNA polymerase to construct DNA replicates of the RNAs;

synthesizing DNA strands complementary to the DNA replicates; and

transcribing the complementary strands using an RNA polymerase that recognizes the promoter region to produce RNA replicates.

32. The method of claim 31 wherein the RNAs comprise mRNAs.

33. The method of claim 32 wherein the mRNAs are obtained from a mammalian tissue.

34. The method of claim 33 wherein the mRNAs are obtained from less than 100 cells.

35. The method of claim 34 wherein the mRNAs are obtained from less than 10 cells.

36. The method of claim 32 wherein the mRNAs is less than 10 ng.

37. The method of claim 33 wherein the tissue is normal.

38. The method of claim 33 wherein the tissue is tumorous or metastatic.

39. The method of claim 31 further comprising storing the solid support for at least 48 hours prior to the transcribing.

40. The method of claim 31 wherein the attached oligonucleotides are the same.

41. The method of claim 31 wherein at least some of the attached oligonucleotides comprise a T7 promoter and a homopolymeric T tract, and a terminal A, G, or C.

42. The method of claim 41 wherein the attached oligonucleotides are covalently attached.

43. The method of claim 41 wherein the attached oligonucleotides are non-covalently attached.

44. The method of claim 31 wherein the RNA replicates are labeled.

45. The method of claim 31 further comprising hybridizing a (labeled) probe to the solid support.

46. The method of claim 31 wherein the solid support is a surface of a well of a multiwell plate.

47. The method of claim 42 wherein the attached oligonucleotides are attached by their 5′

end.

48. The method of claim 46 wherein the solid support is composed of glass.

50. The method of claim 39 wherein the adaptor comprises a promoter region for a second RNA polymerase.

51. The method of claim 40 further comprising reverse transcribing the RNA replicates to form second DNA replicates and transcribing the second DNA replicates using the second RNA polymerase.

52. The method of claim 39 wherein the adaptor further comprises a unique restriction enzyme recognition site, a translational control sequence, or a sequence encoding a purification tag.

49. A method of producing RNA replicates, the method comprising:

providing a solid support having attached oligonucleotides;

annealing a sample that comprises RNAs to the solid support;

extending the attached oligonucleotides using an RNA-directed DNA polymerase to construct DNA replicates of the RNAs;

synthesizing DNA strands complementary to the DNA replicates;

joining an adaptor to the DNA replicates, and transcribing the complementary strand using an RNA polymerase that recognizes the promoter region to produce RNA replicates.

53. A method comprising:

providing a first solid support having 5′

attached oligonucleotide;

annealing a complex sample that comprises sample nucleic acids to the solid support; and

producing template nucleic acids immobilized on the solid support that each include at least a segment of the sample nucleic acids, the immobilized templates representing the composition of the sample nucleic acids.

54. The method of claim 53 wherein the template nucleic acids are archived.

55. The method of claim 53 wherein a master and slave set of template nucleic acids are produced.

56. The method of claim 53 further comprising distributing the template nucleic acids to a user with access to machine-readable information about the composition of the sample nucleic acids.

57. The method of claim 53 wherein the complex sample comprises mRNA from a cell.

58. The method of claim 57 wherein the cell is obtained by microdissection.

59. A method of producing a plurality of dsRNAs, the method comprising:

providing a support comprising a plurality of addresses, each address comprising an immobilized oligonucleotide that includes a first promoter sequence and a target-binding sequence;

contacting each of a plurality of different nucleic acid species to an address of the support under conditions that allow hybridization of each nucleic acid species to the target binding sequence;

synthesizing, at each address, a template nucleic acid that includes the first promoter sequence from the immobilized oligonucleotide, a region of the respective nucleic acid species, and a second promoter sequence, such that the first and second promoter sequences are oriented within the template nucleic acid to transcribe opposing strands of the region;

transcribing the template nucleic acids at each address using one or more RNA polymerases so that complementary transcripts are produced from the template nucleic acid; and

hybridizing the complementary transcripts of each address to each other, thereby providing a dsRNA at each address of the support.