Nucleic acid amplification method: ramification-extension amplification method (RAM)

• US 6,855,523 B2
• Filed: 12/04/2002
• Issued: 02/15/2005
• Est. Priority Date: 06/22/1994
• Status: Expired due to Term

• Current Assignee: Icahn School of Medicine at Mount Sinai
• Original Assignee: Icahn School of Medicine at Mount Sinai
• Inventors: Brandwein, Margaret, Hsuih, Terence C. H., Zhang, David Y.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/309,438
• Publication Number:
  US 20030190604A1
• Time in Patent Office: 804 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 435/91.5, 435/91.51, 435/23.7, 435/23.72, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3, 536/25.32
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

An improved method allowing for rapid sensitive and standardized detection of a target nucleic acid from a pathogenic microorganism or virus or normal or abnormal gene in a sample is provided. The method involves hybridizing a target nucleic acid to several non-overlapping oligonucleotide probes that hybridize to adjacent regions in the target nucleic acid, the probes being referred to capture/amplification probes and amplification probes, respectively, in the presence of paramagnetic beads coated with a ligand binding moiety. Through the binding of a ligand attached to one end of the capture/amplification probe and the specific hybridization of portions of the probes to adjacent sequences in the target nucleic acid, a complex comprising the target nucleic acid, the probes and the paramagnetic beads is formed. The probes may then ligated together to form a contiguous ligated amplification sequence bound to the beads, which complex may be denatured to remove the target nucleic acid and unligated probes. Alternatively, separate capture and amplification probes may be used which form continuous full-length or circular probes, and may be directly detected or amplified using a suitable amplification technique, e.g., PCR, RAM or HSAM for detection. The detection of the ligated amplification sequence, either directly or following amplification of the ligated amplification sequence, indicates the presence of the target nucleic acid in a sample. Methods for the detection of the ligated amplification sequence, including hybridization signal amplification method and ramification-extension amplification method, are also provided.

Claims

1. A method for detecting a target nucleic acid in a sample comprising:

(a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probo comprising a circularizable amplification probe having 3′ and

5′

regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, said 3′ and

5′

regions separated by a genetic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable amplification probe, wherein the circularizable amplification probe is bound on its 3′ and

5′

ends to the adjacent but not the overlapping sequences in the target nucleic acid;

(b) separating the complex from unbound circularizable amplification probes and unbound target nucleic acids and washing the complex;

(c) ligating the 3′ and

5′

ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;

(d) amplifying the circular amplification probe of step (c) by contacting the circular amplification probe of step (c) with a first extension primer that is complementary and hybridizable to generic portions of the circular amplification probe and a second extension primer that is substantially identical to the generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the first extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe, and multiple copies of the second extension primer hybridize to complementary regions of the single stranded DNA and are extended by the DNA polymerase to provide extension products; and

(e) detecting the extension products, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

2. The method of claim 1, wherein the DNA polymerase is Thermus aquaticus (Taq) DNA polymerase.

3. The method of claim 1, wherein the target nucleic acid is specific for a pathogenic microorganism or virus.

4. The method of claim 1, wherein the target nucleic acid is a wild-type or mutant human gene.

5. The method of claim 1, wherein the target nucleic acid is an RNA or DNA molecule.

6. The method of claim 1, wherein the target nucleic acid is selected from the group consisting of HIV-1 RNA, mycobacterial ribosomal RNA, and HCV RNA.

7. The method of claim 6, wherein the ribosomal RNA is 16S rRNA selected from the group consisting of mycobacteria species, M. avium and M. intracellulaire.

8. The method of claim 1, wherein the ligating agent is an enzyme or chemical agent.

9. The method of claim 8, wherein the enzyme is a DNA or RNA ligase.

10. The method of claim 9, wherein the DNA ligase is T4 DNA ligase or Taq DNA ligase.

11. The method of claim 8, wherein the chemical agent is cyanogen bromide or a carbodiimide.

12. The method of claim 1, wherein the sample is a clinical sample.

13. A method for detecting a target nucleic acid in a sample comprising:

(a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequence in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe having 3′ and

5′

regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the 3′ and

5′

regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable amplification probe, wherein the circularizable amplification probe is bound on its 3′ and

5′

ends to the adjacent but not the overlapping sequences in the target nucleic acid;

(b) separating the complex from unbound circularizable amplification probes and unbound target nucleic acids and washing the complex;

(c) ligating the 3′ and

5′

ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;

(d) amplifying the circular amplification probe of step (c) by contacting the circular amplification probe of step (c) with an extension primer that is complementary and hybridizable to generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby said extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe; and

(e) detecting the single stranded DNA, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

14. The method of claim 13, wherein the DNA polymerase is Thermus aquaticus (Taq) DNA polymerase.

15. The method of claim 13, wherein the target nucleic acid is specific for a pathogenic microorganism or virus.

16. The method of claim 13, wherein the target nucleic acid is a wild-type or mutant human gene.

17. The method of claim 13, wherein the target nucleic acid is an RNA or DNA molecule.

18. The method of claim 13, wherein the target nucleic acid is selected from the group consisting of HIV-1 RNA, mycobacterial ribosomal RNA, and HCV RNA.

19. The method of claim 18, wherein the ribosomal RNA is 16S rRNA selected from the group consisting of mycobacteria species, M. avium and M. intracellulaire.

20. The method of claim 13, wherein the ligating agent is an enzyme or chemical agent.

21. The method of claim 20, wherein the enzyme is a DNA or RNA ligase.

22. The method of claim 21, wherein the DNA ligase is T4 DNA ligase or Taq DNA ligase.

23. The method of claim 20, wherein the chemical agent is cyanogen bromide or a carbodiimide.

24. The method of claim 13, wherein the sample is a clinical sample.

25. A method for detecting a target nucleic acid in a sample comprising:

(a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe having 3′ and

5′

regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, said 3′ and

5′

regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable amplification probe, wherein the circularizable amplification probe is bound on its 3′ and

5′

ends to the adjacent but not the overlapping sequences in the target nucleic acid;

(b) ligating the 3′ and

5′

ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;

(c) amplifying the circular amplification probe of step (b) by contacting the circular amplification probe of step (b) with a first extension primer that is complementary and hybridizable to generic portions of the circular amplification probe and a second extension primer that is substantially identical to the generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby the first extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe, and multiple copies of the second extension primer hybridize to complementary regions of the single stranded DNA and are extended by the DNA polymerase to provide extension products; and

(d) detecting the extension products, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

26. The method of claim 25, wherein the DNA polymerase is Thermus aquaticus (Taq) DNA polymerase.

27. The method of claim 25, wherein the target nucleic acid is specific for a pathogenic microorganism or virus.

28. The method of claim 25, wherein the target nucleic acid is a wild-type or mutant human gene.

29. The method of claim 25, wherein the target nucleic acid is an RNA or DNA molecule.

30. The method of claim 25, wherein the target nucleic acid is selected from the group consisting of HIV-1 RNA, mycobacterial ribosomal RNA, and HCV RNA.

31. The method of claim 30, wherein the ribosomal RNA is 16S rRNA selected from the group consisting of mycobacteria species, M. avium and M. intracellulaire.

32. The method of claim 25, wherein the ligating agent is an enzyme or chemical agent.

33. The method of claim 32, wherein the enzyme is a DNA or RNA ligase.

34. The method of claim 33, wherein the DNA ligase is T4 DNA ligase or Taq DNA ligase.

35. The method of claim 32, wherein the chemical agent is cyanogen bromide or a carbodiimide.

36. The method of claim 25, wherein the sample is a clinical sample.

37. A method for detecting a target nucleic acid in a sample comprising:

(a) contacting the nucleic acid in the sample under conditions that allow nucleic acid hybridization between complementary sequences in the nucleic acid with at least one oligonucleotide probe, the oligonucleotide probe comprising a circularizable amplification probe having 3′ and

5′

regions that are complementary to adjacent but not overlapping sequences in the target nucleic acid, the 3′ and

5′

regions separated by a generic region that is neither complementary nor hybridizable to a nucleotide sequence in the target nucleic acid, such that a complex is formed comprising the target nucleic acid and the circularizable amplification probe, wherein the circularizable amplification probe is bound on its 3′ and

5′

ends to the adjacent but not the overlapping sequences in the target nucleic acid;

(b) ligating the 3′ and

5′

ends of the circularizable amplification probe with a ligating agent that joins nucleotide sequences such that a circular amplification probe is formed;

(c) amplifying the circular amplification probe of step (b) by contacting the circular amplification probe of step (b) with an extension primer that is complementary and hybridizable to generic portions of the circular amplification probe, dNTPs, and a DNA polymerase having strand displacement activity, under conditions whereby said extension primer is extended around the circular amplification probe for multiple revolutions to form a single stranded DNA of repeating units complementary to the sequence of the circular amplification probe; and

(d) detecting the single stranded DNA, wherein detection thereof indicates the presence of the target nucleic acid in the sample.

38. The method of claim 37, wherein the DNA polymerase is Thermus aquaticus (Taq) DNA polymerase.

39. The method of claim 37, wherein the target nucleic acid is specific for a pathogenic microorganism or virus.

40. The method of claim 37, wherein the target nucleic acid is a wild-type or mutant human gene.

41. The method of claim 37, wherein the target nucleic acid is an RNA or DNA molecule.

42. The method of claim 37, wherein the target nucleic acid is selected from the group consisting of HIV-1 RNA, mycobacterial ribosomal RNA, and HCV RNA.

43. The method of claim 42, wherein the ribosomal RNA is 16S rRNA selected from the group consisting of mycobacteria species, M. avium and M. intracellulaire.

44. The method of claim 37, wherein the ligating agent is an enzyme or chemical agent.

45. The method of claim 44, wherein the enzyme is a DNA or RNA ligase.

46. The method of claim 45, wherein the DNA ligase is T4 DNA ligase or Taq DNA ligase.

47. The method of claim 44, wherein the chemical agent is cyanogen bromide or a carbodiimide.

48. The method of claim 37, wherein the sample is a clinical sample.