Detecting the presence of specific target nucleic acid sequences through stem-loop formation

• US 6,764,821 B1
• Filed: 11/12/1999
• Issued: 07/20/2004
• Est. Priority Date: 06/24/1998
• Status: Expired due to Term

• Current Assignee: Enzo Diagnostics, Inc.
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Stavrianopoulos, Jannis G., Donegan, James J., Rabbani, Elazar, Coleman, Jack, Walner, Marleen
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/439,594
• Time in Patent Office: 1,712 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 536/25.32
• US Class Current: 435/6.16
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]

Abstract

This invention provides novel processes for amplifying nucleic acid sequences of interest, including linear and non-linear amplification. In linear amplification, a single initial primer or nucleic acid construct is utilized to carry out the amplification process. In nonlinear amplification, a first initial primer or nucleic acid construct is employed with a subsequent initial primer or nucleic acid construct. In other non-linear amplification processes provided by this invention, a first initial primer or nucleic acid construct is deployed with a second initial primer or nucleic acid construct to amplify the specific nucleic acid sequence of interest and its complement that are provided. A singular primer or a singular nucleic acid construct capable of non-linear amplification can also be used to carry out non-linear amplification in accordance with this invention. Post-terminatlon labeling process for nucleic acid sequencing is also disclosed in this invention that is based upon the detection of tagged molecules that are covalently bound to chemically reactive groups provided for chain terminators. A process for producing nucleic acid sequences having decreased thermodynamic stability to complementary sequences is also provided and achieved by this invention. Unique nucleic acid polymers are also disclosed and provided in addition to other novel compositions, kits and the like.

Claims

1. A process for detecting the presence of a specific target nucleic acid sequence comprising the steps of:

1) providing one or more first initial primers or first nucleic acid constructs comprising two segments;

(A) a first segment (i) being substantially complementary to a portion of said specific target nucleic acid sequence and (ii) which provides for template dependent extension; and

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) substantially complementary to sequences that are synthesized by extension of the first segment of said first initial primers or first nucleic acid constructs with said specific target nucleic acid sequences as a template and where in the absence of a denaturation step, the first initial primers or first nucleic acid constructs participate in the formation of a stem-loop structure after said specific target nucleic acid sequence is used as a template for extension;

2) mixing said first initial primer or nucleic acid constructs with substrates, buffer, a template-dependent polymerizing enzyme and a sample to be tested for the presence of said specific target nucleic acid;

3) incubating said mixture under temperature conditions sufficient for binding of said first initial primers or first nucleic acid constructs to said specific target nucleic acid sequence, extension of said first initial primers or first nucleic acid constructs;

4) forming at least one stem-loop structure by

(a) self-annealing between said second segment of said first initial primer or first nucleic acid construct and a segment formed after target template dependent extension of the first segment of said first initial primer or first nucleic acid construct, and

(b) separating said first segment of said first initial primer or first nucleic acid construct from said specific target nucleic acid sequence, and

5) detecting the presence of said stem-loop structures formed in said forming step (4), thereby detecting the presence of said specific target nucleic acid sequence or its complement.

2. The process of claim 1, wherein prior to or after said mixing step 2) any double-stranded nucleic acids in said sample have been rendered single-stranded.

3. The process of claim 1, wherein said single-stranded rendering has been carried out by heat denaturation.

4. The process of claim 1, wherein said template polymerizing enzyme comprises a member selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase and a combination of any of the foregoing.

5. The process of claim 1, wherein said first initial primers, extended first initial primers, first nucleic acid constructs or extended first nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

6. The process of claim 1, wherein said first initial primer or first nucleic acid construct or said extended first initial primer or first nucleic acid construct comprises one or more modified nucleotides having detectable labels.

7. The process of claim 5 or 6, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

8. The process of any of claim 1, 2, 3, 4, 5 or 6, wherein said mixing step 2), the sample is the product of an amplification reaction.

9. The process of any of claim 1, 2, 3, 4, 5, or 6, wherein said mixing step 2), the sample has not previously undergone amplification or has been subjected to an amplification process.

10. The process of claim 7, wherein said mixing step 2), the sample is the product of an amplification reaction.

11. The process of claim 7, wherein said mixing step 2), the sample has not previously undergone amplification or has been subjected to an amplification process.

12. The process of claim 1, wherein said providing step 1) at least one second initial primer or second nucleic acid construct is provided, wherein said second initial primer or second nucleic acid construct comprises a first segment substantially complementary to sequences that are synthesized after extension of said first initial primer or first nucleic acid construct.

13. The process of claim 2, wherein said second initial primer or second nucleic acid construct further comprises a second segment being (i) substantially non-identical to the first segment of said second initial primer or second nucleic acid construct;

(ii) substantially identical to a portion of said extended first initial primer or first nucleic acid construct; and

(iii) substantially complementary to sequences that are synthesized by extension of the first segment of said second initial primer or second nucleic acid construct with said extended first primer or nucleic acid construct as a template.

14. The process of claim 13, wherein said separating step 4)(b) is carried out by strand displacement, said strand displacement being carried out by extending at least one additional primer that binds to a site closer to the 3′

end of said specific target nucleic acid sequence than the binding site for a first segment of a first initial primer or first nucleic acid construct or a second initial primer or second nucleic acid construct.

15. The process of claim 12, wherein said second initial primers, extended initial primers, nucleic acid constructs or extended nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

16. The process of claim 13 or 14, wherein said second initial primer or second nucleic acid construct or said extended second initial primer or extended second nucleic acid construct comprises one or more modified nucleotides having detectable labels.

17. The process of claims 15, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

18. The process of claim 16, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

19. The process of claim 1, 12 or 13, wherein the detection of the formation of the stem-loop structure takes place after nucleic acid synthesis is substantially completed.

20. The process of claim 1, 12 or 13, wherein the detection of the formation of the stem-loop structure takes place before nucleic acid synthesis is substantially completed.

21. A process for detecting the presence of a specific target nucleic acid sequence comprising the steps of:

1) providing one or more first initial primers or first nucleic acid constructs comprising two segments;

(A) a first segment (i) being substantially complementary to a portion of said specific target nucleic acid sequence and (ii) which provides for template dependent extension; and

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) substantially complementary to sequences that are synthesized by extension of the first segment of said first initial primers or first nucleic acid constructs with said specific target nucleic acid sequences as a template and wherein the first initial primers or first nucleic acid constructs participate in the formation of a stem-loop structure after said specific target nucleic acid sequence is used as a template for extension;

2) mixing said first initial primer or nucleic acid constructs with substrates, buffer, a template-dependent polymerizing enzyme and a sample to be tested for the presence of said specific target nucleic acid;

3) incubating said mixture under temperature conditions sufficient for binding of said first initial primers or first nucleic acid constructs to said specific target nucleic acid sequence, extension of said first initial primers or first nucleic acid constructs, and wherein said extension is only carried out using first initial primers or nucleic acid constructs comprising said two segments;

4) forming at least one stem-loop structure by

(a) self-annealing between said second segment of said first initial primer or first nucleic acid construct and a segment formed after target template dependent extension of the first segment of said first initial primer or first nucleic acid construct, and

(b) separating said first segment of said first initial primer or first nucleic acid construct from said specific target nucleic acid sequence, and

5) detecting the presence of said stem-loop structures formed in said forming step (4), thereby detecting the presence of said specific target nucleic acid sequence or its complement.

22. The process of claim 21, wherein prior to or after said mixing step 2) any double-stranded nucleic acids in said sample have been rendered single-stranded.

23. The process of claim 21, wherein said single-stranded rendering has been carried out by heat denaturation.

24. The process of claim 21, wherein said template polymerizing enzyme comprises a member selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase and a combination of any of the foregoing.

25. The process of claim 21, wherein said first initial primers, extended first initial primers, first nucleic acid constructs or extended first nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

26. The process of claim 21, wherein said first initial primer or first nucleic acid construct or said extended first initial primer or first nucleic acid construct comprises one or more modified nucleotides having detectable labels.

27. The process of claim 25 or 26, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

28. The process of any of claim 21, 22, 23, 24, 25 or 26, wherein said mixing step 2), the sample is the product of an amplification reaction.

29. The process of any of claim 27, wherein said mixing step 2), the sample has not previously undergone amplification or has been subjected to an amplification process.

30. The process of claim 21, wherein said providing step 1) at least one second initial primer or second nucleic acid construct is provided, wherein said second initial primer or second nucleic acid construct comprises a first segment substantially complementary to sequences that are synthesized after extension of said first initial primer or first nucleic acid construct and is substantially non-identical to any sequences in said first initial primer or first nucleic acid construct.

31. The process of claim 30, wherein said second initial primer or second nucleic acid construct further comprises a second segment being (i) substantially non-identical to the first segment of said second initial primer or second nucleic acid construct;

(ii) substantially identical to a portion of said extended first initial primer or first nucleic acid construct; and

(iii) substantially complementary to sequences that are synthesized by extension of the first segment of said second initial primer or second nucleic acid construct with said extended first primer or nucleic acid construct as a template.

32. The process of claim 21, wherein said separating step 4)(b) is carried out by strand displacement, said strand displacement being carried out by extending at least one additional primer that binds to a site closer to the 3′

end of said specific target nucleic acid sequence than the binding site for a first segment of a first initial primer or first nucleic acid construct or a second initial primer or second nucleic acid construct.

33. The process of claim 30, wherein said second initial primers, extended initial primers, nucleic acid constructs or extended nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

34. The process of claim 30, wherein said second initial primer or second nucleic acid construct or said extended second initial primer or extended second nucleic acid construct comprises one or more modified nucleotides having detectable labels.

35. The process of claim 34, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

36. The process of any of claim 21, 25, 26, 30, 31, 32, 33, 34 or 35, wherein the detection of the formation of the stem-loop structure takes place after nucleic acid synthesis is substantially completed.

37. The process of any of claim 21, 25, 26, 30, 31, 32, 33, 34 or 35, wherein the detection of the formation of the stem-loop structure takes place before nucleic acid synthesis is substantially completed.

38. A process for detecting the presence of a specific target nucleic acid sequence comprising the steps of:

1) providing one or more first initial primers or first nucleic acid constructs comprising two segments;

(A) a first segment (i) being substantially complementary to a portion of said specific target nucleic acid sequence and (ii) which provides for template dependent extension; and

(B) a second segment being (i) substantially non-identical to said first segment, (ii) substantially identical to a portion of said specific target nucleic acid sequence, (iii) substantially complementary to sequences that are synthesized by extension of the first segment of said first initial primers or first nucleic acid constructs with said specific target nucleic acid sequences as a template to form a first extended nucleic acid strand, and wherein the first initial primers or first nucleic acid constructs being participate in the formation of a stem-loop structure at one end of said first extended nucleic acid strand after said specific target nucleic acid sequence is used as a template for extension;

2) mixing said first initial primer or nucleic acid constructs with substrates, buffer, a template-dependent polymerizing enzyme and a sample to be tested for the presence of said specific target nucleic acid;

3) incubating said mixture under temperature conditions sufficient for binding of said first initial primers or first nucleic acid constructs to said specific target nucleic acid sequence, extension of said first initial primers or first nucleic acid constructs, and wherein said extension is only carried out using first initial primers or nucleic acid constructs comprising said two segments;

4) forming at least one stem-loop structure by

(a) self-annealing between said second segment of said first initial primer or first nucleic acid construct and a segment formed after target template dependent extension of the first segment of said first initial primer or first nucleic acid construct, and

b) separating said first segment of said first initial primer or first nucleic acid construct from said specific target nucleic acid sequence, and

5) detecting the presence of said stem-loop structures formed in said forming step (4), thereby detecting the presence of said specific target nucleic acid sequence or its complement.

39. The process of claim 38, wherein prior to or after said mixing step 2) any double-stranded nucleic acids in said sample have been rendered single-stranded.

40. The process of claim 38, wherein said single-stranded rendering has been carried out by heat denaturation.

41. The process of claim 38, wherein said template polymerizing enzyme comprises a member selected from the group consisting of DNA polymerase, RNA polymerase, reverse transcriptase, DNA ligase and a combination of any of the foregoing.

42. The process of claim 38, wherein said first initial primers, extended first initial primers, first nucleic acid constructs or extended first nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

43. The process of claim 38, wherein said first initial primer or first nucleic acid construct or said extended first initial primer or first nucleic acid construct comprises one or more modified nucleotides having detectable labels.

44. The process of claim 42, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

45. The process of claim 43, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

46. The process of any of claim 38, 42, 43, 44 or 45, wherein said mixing step 2), the sample is the product of an amplification reaction.

47. The process of any of claim 38, 42, 43, 44 or 45, wherein said mixing step 2), the sample has not previously undergone amplification or has been subjected to an amplification process.

48. The process of claim 38, wherein said providing step 1) at least one second initial primer or second nucleic acid construct is provided, wherein said second initial primer or second nucleic acid construct comprises a first segment substantially complementary to sequences that are synthesized after extension of said first initial primer or first nucleic acid construct.

49. The process of claim 48, wherein said second initial primer or second nucleic acid construct further comprises a second segment being (i) substantially non-identical to the first segment of said second initial primer or second nucleic acid construct;

(ii) substantially identical to a portion of said extended first initial primer or first nucleic acid construct; and

(iii) substantially complementary to sequences that are synthesized by extension of the first segment of said second initial primer or second nucleic acid construct with said extended first primer or nucleic acid construct as a template.

50. The process of claim 38, wherein said separating step 4) (b) is carried out by strand displacement, said strand displacement being carried out by extending at least one additional primer that binds to a site closer to the 3′

end of said specific target nucleic acid sequence than the binding site for a first segment of a first initial primer or first nucleic acid construct or a second initial primer or second nucleic acid construct.

51. The process of claim 48, wherein said second initial primers, extended initial primers, nucleic acid constructs or extended nucleic acid constructs comprise one or more moieties selected from the group consisting of modified nucleotides, non-nucleic acid polymers, branched nucleic acids, inverted nucleic acids, peptide nucleic acids, spacer groups, abasic sites, detectable labels and a combination of any of the foregoing.

52. The process of claim 48, wherein said second initial primer or second nucleic acid construct or said extended second initial primer or extended second nucleic acid construct comprises one or more modified nucleotides having detectable labels.

53. The process of claim 48, wherein said detectable labels are selected from the group consisting of haptens, ligands, receptors, fluorescein, rhodamine, coumarin, fluoresceinated molecules, infra-rad fluorescent groups, chemiluminescent moieties, energy transfer systems, enzymes and a combination of any of the foregoing.

54. The process of any of claim 38, 42, 43, 44, 45, 48, 49, 50, 51, 52 or 53, wherein the detection of the formation of the stem-loop structure takes place after nucleic acid synthesis is substantially completed.

55. The process of any of claim 38, 42, 43, 44, 45, 48, 49, 50, 51, 52, or 53 wherein the detection of the formation of the stem-loop structure takes place before nucleic acid synthesis is substantially completed.