Methods and compositions for transcription-based nucleic acid amplification

• US 6,686,156 B2
• Filed: 06/26/2001
• Issued: 02/03/2004
• Est. Priority Date: 06/26/2000
• Status: Expired due to Fees

• Current Assignee: Elizabeth Davila, Juan Davila, NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Kurn, Nurith
• Primary Examiner(s): Forman, B. J.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/893,191
• Publication Number:
  US 20020058270A1
• Time in Patent Office: 952 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• 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 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/143 incorporating a promoter sequence
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

Methods for isothermal exponential amplification of a target polynucleotide are disclosed. The methods employ two transcription modules, the first module providing linear amplification resulting in RNA transcripts, and a second module providing for further (generally cyclical) amplification resulting in more RNA transcripts. In one aspect, the amplification of the first module is composite primer based. In a second aspect, the amplification of the first module is based on target switching to generate a primer extension product comprising a promoter sequence. In all aspects, the RNA transcripts of the first transcription module are subjected to further amplification by creating an intermediate product comprising a double stranded promoter region from which transcription can occur. The invention further provides compositions and kits for practicing said methods, as well as methods which use the amplification results.

Claims

1. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) hybridizing a target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the target polynucleotide is single-stranded;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide (4 sequence to a region of the target polynucleotide 5′

with respect to a site for hybridization of the first primer to the target polynucleotide;

(c) extending the first primer with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide;

(d) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;

(e) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising a sequence complementary to the displaced primer extension products is produced;

(f) hybridizing a second primer to the RNA transcript of step (e);

(g) extending the second primer with an enzyme comprising RNA-dependent DNA polymerase activity to generate a complex comprising a second primer extension product and the RNA transcript;

(h) cleaving RNA in the complex of step (g) with an enzyme that cleaves RNA in an RNA/DNA hybrid;

(i) hybridizing the second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising the nucleic acid sequence of interest is produced;

whereby multiple copies of the nucleic acid sequence of interest are produced.

2. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining;

the complex of step (c) of claim 1;

a composite primer that is hybridizable to the target polynucleotide, wherein the composite primer comprises an RNA portion and a 3′

DNA portion;

an enzyme that cleaves RNA from an RNA/DNA hybrid;

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to displaced composite primer extension product;

an RNA polymerase;

a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;

an enzyme comprising RNA-dependent DNA polymerase activity; and

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and

(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, displacement of the first primer extension product from the complex of step (c) of claim 1 when its RNA is cleaved and a composite primer binds to the target polynucleotide in the complex, hybridization of a propromoter polynucleotide to a first primer extension product to form a complex comprising a first primer extension product and a propromoter polynucleotide, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a second primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

3. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining;

the displaced primer extension product of step (d) of claim 1;

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to displaced first primer extension product;

an RNA polymerase;

a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;

an enzyme comprising RNA-dependent DNA polymerase activity;

an enzyme that cleaves RNA from an RNA/DNA hybrid; and

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and

(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a first primer extension product to form a complex comprising a first primer extension product and a propromoter polynucleotide, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a second primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

6. The method of any of claims 1-3 or 5, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

7. The method of claim 6, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

8. The method of claim 1 or 5, wherein a plurality of composite primers are used.

9. The method of claim 1 or 5, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).

10. The method of claim 9, wherein the TSO comprises a modification in the region which hybridizes to the target polynucleotide, wherein, under a given set of conditions, the TSO binds more tightly to the region as compared to a TSO without the modification.

11. The method of claim 1 or 5, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

12. The method of claim 11, wherein the blocking sequence comprises a modification in the region which hybridizes to the target polynucleotide, wherein, under a given set of conditions, the blocking sequence binds more tightly to the region as compared to a blocking sequence without the modification.

13. The method of claim 1 or 5, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

14. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a displaced first primer extension product is a template switch oligonucleotide (TSO).

15. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded second primer extension product is a TSO.

16. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a displaced first primer extension product is a propromoter template oligonucleotide (PTO).

17. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded second primer extension product is a PTO.

18. The method of claim 1 or 5, wherein at least one type of rNTP used is a labeled rNTP, whereby labeled RNA products are generated.

19. The method of claim 1 or 5, wherein a single enzyme comprises RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

20. The method of claim 1 or 5, wherein a single enzyme comprises RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

21. The method of claim 1 or 5, wherein a single enzyme comprises DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleaves RNA from an RNA/DNA hybrid.

22. A method of sequencing a target polynucleotide, said method comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1 in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog;

and (b) analyzing the amplification products to determine sequence.

23. A method of sequencing a target polynucleotide, said method comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1, wherein RNA transcripts generated from a first primer extension product are amplified in the presence of a mixture of rNTPs and rNTP analogs such that transcription is terminated upon incorporation of an rNTP analog;

and (b) analyzing the amplification products to determine sequence.

24. A method of detecting a mutation in a target polynucleotide by single stranded conformation polymorphism, comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1;

and (b) analyzing the amplification products for single stranded conformation, wherein a difference in conformation as compared to a reference single stranded polynucleotide indicates a mutation in the target polynucleotide.

25. A method of characterizing a sequence of interest in a target polynucleotide, said method comprising

(i) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1, wherein the sequence of the RNA portion of the composite primer is known, and

(ii) comparing the amplification products if any from step (i) with the amount of amplification products from a reference template

wherein

(1) production of detectably fewer amplification products from the target polynucleotide as compared to the amount of amplification products from the reference template which comprises, a region complementary to the RNA portion of the composite primer indicates that the target polynucleotide does not comprise a sequence complementary to the RNA portion of the composite primer and is a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer;

or

(2) production of detectably more amplification products from the target polynucleotide as compared to the amount of amplification products from the reference template which does not comprise a region which is complementary to the RNA portion of the composite primer indicates that the target polynucleotide comprises a sequence complementary to the RNA portion of the composite primer and is not a sequence variant with respect to the sequence complementary to the RNA portion of the composite primer.

26. The method of claim 25, wherein the sequence of the RNA portion of the composite primer comprises a sequence complementary to a wild type sequence.

27. The method of claim 25, wherein the sequence of the RNA portion of the composite primer comprises a sequence complementary to a mutant sequence.

28. A method of producing a microarray, comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1;

and (b) immobilizing the amplification products on a substrate to fabricate a microarray comprising the amplification products.

29. A method of characterizing a sequence of interest, comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 18;

and (b) analyzing the labeled RNA products.

30. The method of claim 29, wherein step (b) comprises contacting the labeled RNA products with at least one probe.

31. The method of claim 30, wherein the at least one probe is provided as a microarray.

32. The method of claim 31, wherein the microarray comprises at least one probe immobilized on a substrate fabricated from a material selected from the group consisting of paper, glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, and optical fiber.

33. The method of claim 32, wherein the substrate comprises pins, rods, fibers, tapes, threads, beads, particles, microtiter wells, capillaries, or cylinders.

34. The method of claim 29, wherein step (b) of analyzing the labeled RNA products comprises determining amount of said labeled RNA products, whereby the amount of the sequence of interest present in a sample is quantified.

35. A method of determining gene expression profile in a sample, said method comprising:

(a) generating multiple Copies of at least two nucleic acid sequences of interest in the sample using the method of claim 1; and

(b) determining amount of amplification products of each sequence of interest, wherein each said amount is indicative of amount of each sequence of interest in the sample, whereby the gene expression profile in the sample is determined.

36. The method of claim 35, wherein the at least two sequences of interest comprise cDNA.

37. A method of determining sequence of a sequence of interest comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1;

and (b) sequencing the amplification products.

38. A method of detecting presence of a nucleic acid sequence of interest in a sample, said method comprising (a) generating multiple copies of a nucleic acid sequence of interest by the method of claim 1;

and (b) detecting presence or absence of the sequence of interest in the amplification products, if any.

39. The method of claim 7, wherein the RNA portion of the composite primer consists of 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

40. The method of claim 7, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

41. The method of claim 1 or 5, wherein different enzymes comprise RNA-dependent DNA polymerase activity and DNA-dependent DNA polymerase activity.

42. The method of claim 1 or 5, wherein different enzymes comprise RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

43. The method of claim 1 or 5, wherein different enzymes comprise DNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

44. The method of claim 1 or 5, wherein different enzymes comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity and cleave RNA from an RNA/DNA hybrid.

54. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a displaced first primer extension product is a propromoter template oligonucleotide (PTO).

55. The method of claim 1 or 5, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded second primer extension product is a PTO.

57. The method of any of claims 22-25, 28, 35, 37, 38, or 56, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

58. The method of claim 57, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

59. The method of claim 1, wherein the method comprises hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template which is 5′

with respect to hybridization of the composite primer to the template.

61. The method of claim 29, wherein the RNA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

4. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining;

an RNA transcript, wherein said RNA transcript is produced by a method comprising the steps of;

(i) hybridizing a target polynucleotide comprising the nucleic acid sequence of interest with a first primer, wherein said first primer is a composite primer comprising an RNA portion and a 3′

DNA portion, wherein the target polynucleotide is single-stranded;

(ii) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5′

with respect to a site for hybridization of the first primer to the target polynucleotide;

(iii) extending the first primer with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide;

(iv) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;

(v) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising a sequence complementary to the displaced primer extension products is produced;

a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;

an enzyme comprising RNA-dependent DNA polymerase activity;

an enzyme that cleaves RNA from an RNA/DNA hybrid;

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and

an RNA polymerase; and

(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, hybridization of a propromoter polynucleotide to a primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

45. The method of claim 4, wherein the IRMA portion of the composite primer is 5′

with respect to the 3′

DNA portion.

46. The method of claim 45, wherein the 5′

RNA portion is adjacent to the 3′

DNA portion.

47. The method of claim 46, wherein the RNA portion of the composite primer consists of 7 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

48. The method of claim 46, wherein the RNA portion of the composite primer consists of about 10 to about 20 nucleotides and the DNA portion of the composite primer consists of about 5 to about 20 nucleotides.

49. The method of claim 4, wherein the enzyme that cleaves RNA from an RNA/DNA hybrid is RNaseH.

50. The method of claim 4, wherein the polynucleotide comprising a termination polynucleotide sequence is a template switch oligonucleotide (TSO).

51. The method of claim 4, wherein the polynucleotide comprising a termination polynucleotide sequence is a blocking sequence.

52. The method of claim 4, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a displaced first primer extension product is a template switch oligonucleotide (TSO).

53. The method of claim 4, wherein the propromoter polynucleotide comprising a propromoter and a region which hybridizes to a single stranded second primer extension product is a TSO.

5. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) combining;

a first primer, wherein the first primer is a composite primer that is hybridizable to a target polynucleotide, and wherein the composite primer comprises an RNA portion and a 3′

DNA portion;

optionally a polynucleotide comprising a termination polynucleotide sequence that is hybridizable to a region of the target polynucleotide which is 5′

with respect to hybridization of the composite primer to the target polynucleotide;

an enzyme comprising DNA-dependent DNA polymerase activity;

an enzyme that cleaves RNA from an RNA/DNA hybrid;

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a first primer extension product;

an RNA polymerase;

a second primer that is hybridizable to a sense RNA transcript comprising the nucleic acid sequence of interest;

an enzyme comprising RNA-dependent DNA polymerase activity; and

a propromoter polynucleotide comprising a propromoter and a region which hybridizes to a second primer extension product; and

(b) incubating the mixture of step (a) under conditions that permit primer hybridization and extension, RNA cleavage, displacement of a first primer extension product from a complex comprising a first primer extension product and target polynucleotide when its RNA is cleaved and a composite primer binds to the target polynucleotide in the complex, hybridization of a propromoter polynucleotide to a second primer extension product to form a complex comprising a primer extension product and a propromoter polynucleotide, and RNA transcription, whereby multiple copies of the nucleic acid sequence of interest are generated.

60. The method of claim 5, wherein step (a) of combining comprises:

a polynucleotide comprising a termination polynucleotide sequence that is hybridizable to a region of the target polynucleotide which is 5′

with respect to hybridization of the composite primer to the target polynucleotide.

56. A method of generating multiple copies of a nucleic acid sequence of interest, said method comprising the steps of:

(a) extending a first primer hybridized to a target polynucleotide comprising the nucleic acid sequence of interest with an enzyme comprising DNA-dependent DNA polymerase activity to generate a complex comprising a first primer extension product and target polynucleotide, wherein said first primer is a composite primer comprising an RNA portion and a 3′

DNA portion;

(b) optionally hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the target polynucleotide 5′

with respect to a site for hybridization of the first primer to the target polynucleotide;

(c) cleaving the RNA portion from the composite primer in the complex of first primer extension product and target polynucleotide with an enzyme that cleaves RNA from an RNA/DNA hybrid such that another composite primer can hybridize to the target polynucleotide and repeating primer extension by strand displacement to produce a displaced primer extension product;

(e) hybridizing a propromoter polynucleotide comprising a propromoter and a region which hybridizes to the displaced primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising sequences complementary to the displaced primer extension products is produced;

(f) generating a second primer extension product complementary to the RNA transcript;

(g) hybridizing the second primer extension product with a propromoter polynucleotide, wherein the propromoter polynucleotide comprises a propromoter and a region which hybridizes to the single stranded second primer extension product under conditions which allow transcription to occur by RNA polymerase, such that an RNA transcript comprising the nucleic acid sequence of interest is produced;

whereby multiple copies of the nucleic acid sequence of interest are produced.