Self initiating single primer amplification of nucleic acids

• US 6,294,323 B1
• Filed: 04/14/1993
• Issued: 09/25/2001
• Est. Priority Date: 04/14/1993
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behringwerke Aktiengesellschaft (Sanofi )
• Inventors: Rose, Samuel J., Ullman, Edwin F.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Tran, Paul B.
• Application Number:
  US08/046,682
• Time in Patent Office: 3,086 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/810, 435/91.5, 435/15, 935/77, 935/78, 935/91.1, 536/24.33
• US Class Current: 435/6.12
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released

Abstract

A method is disclosed for producing at least one copy of a pair of complementary single stranded polynucleotides. The method comprises forming, in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along each of the complementary single stranded polynucleotides, an extension of a polynucleotide primer. The polynucleotide primer is comprised of at least a sequence of 16 nucleotides terminating at its 3′ end in a 2 to 9 nucleotide sequence (S1), which is complementary with the 3′ ends of both of the complementary single stranded polynucleotides. The polynucleotide primer has at least an 8 nucleotide sequence (S2) that is 5′ of S1, where S2 is 50 to 80% complementary to the nucleotide sequences contiguous with the 3′ ends of the complementary single stranded polynucleotides. The extended polynucleotide primer and the single stranded polynucleotides are then dissociated.

Claims

1. A method of producing at least one copy of a pair of complementary single stranded polynucleotide sequences, said method comprising:

(a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase along each of said complementary single stranded polynucleotide sequences, an extension of a single polynucleotide primer, said polynucleotide primer being comprised of at least a sequence of 16 nucleotides terminating at its 3′

end in a 4 to 8 nucleotide sequence (S1) that is complementary with the 3′

ends of both of said complementary single stranded polynucleotide sequences, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′

of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′

ends of said complementary single stranded polynucleotide sequences, and

(b) dissociating said extended polynucleotide primer and said single stranded polynucleotides thereby producing said copy of said single stranded polynucleotide sequences.

2. The method of claim 1 wherein said S2 is at least 60% complementary with each of said nucleotide sequences contiguous with the 3′

ends of said complementary single stranded polynucleotide sequences.

3. The method of claim 1 wherein said pair of single stranded polynucleotide sequences and said copy are DNA.

4. The method of claim 1 wherein said polynucleotide primer is 24 to 90 nucleotides in length.

5. The method of claim 1 wherein said S2 contains at least one ambiguous nucleotide capable of binding to A, T, G and C.

6. The method of claim 5 wherein said ambiguous nucleotide is inosine.

7. The method of claim 1 wherein said S1 is at least 75% G and C.

8. The method of claim 1 wherein said S2 comprises a series of multiplets of nucleotides, said multiplets taken in order corresponding to at least 50% complementary multiplets at respectively ordered positions located alternately within sequences contiguous with the 3′

ends of each of said complementary single stranded polynucleotide sequences.

9. The method of claim 8 wherein said S2 consists solely of said multiplets.

10. The method of claim 8 wherein said multiplets are quartets.

11. The method of claim 1 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

12. The method of claim 1 wherein said method is carried out at at least a 100-fold excess concentration of said polynucleotide primer relative to the concentration of said single stranded polynucleotides.

13. A method comprising repetition of the steps of claim 1 wherein said copy of said pair of complementary single stranded polynucleotide sequences has bonded to each of its 3′

ends a sequence that is identical to said sequence S2 and a sequence at each of the 5′

ends that is complementary to said sequence S2.

14. A method comprising repetition of the steps of claim 1 wherein the number of copies of said pair of single stranded polynucleotide sequences is increased by at least a factor of a thousand.

15. A method of producing multiple copies of a polynucleotide sequence and its complement, which comprises:

(a) providing in combination (1) a pair of complementary single stranded polynucleotides having said polynucleotide sequence and its complement, (2) a single polynucleotide primer being comprised of a sequence of 24 to 90 nucleotides terminating at its 3′

end in a 2 to 9 nucleotide sequence (S1) that is complementary with the 3′

ends of both of said polynucleotide sequences and its complement, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′

of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′

ends of said polynucleotide sequence and its complement, (3) nucleoside triphosphates, (4) template dependent polynucleotide polymerase and

(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating said polynucleotide sequence and its complement, (2) hybridizing said polynucleotide primer with the sequences at the 3′

end of said polynucleotide sequence and its complement, (3) extending said polynucleotide primer along said polynucleotide sequence and its complement to provide a first complementary pair of extended polynucleotide primers, (4) dissociating said first complementary pair of extended polynucleotide primers from said polynucleotide sequence and its complement, (5) hybridizing single stranded extended polynucleotide primers from said first complementary pair with said polynucleotide primer, (6) extending said polynucleotide primer along said single stranded extended polynucleotide primers to provide a second complementary pair of extended polynucleotide primers, (7) dissociating said second complementary pair of extended polynucleotide primers from said first complementary pair of extended polynucleotide primers, and (8) repeating steps (5)-(7) above thereby producing multiple copies of said polynucleotide sequences.

16. The method of claim 15 wherein said S1 is 4 to 8 nucleotides in length.

17. The method of claim 15 wherein said S2 is at least 60% complementary with each of said nucleotide sequences contiguous with the 3′

ends of said polynucleotide sequence and its complement.

18. The method of claim 15 wherein said polynucleotide sequence or its complement or said first or second extended polynucleotide primer is DNA.

19. The method of claim 15 wherein said S2 contains at least one ambiguous nucleotide capable of binding to A, T, G and C.

20. The method of claim 19 wherein said ambiguous nucleotide is inosine.

21. The method of claim 15 wherein said S2 contains 1 to 4 ambiguous nucleotides capable of binding to A, T, G and C.

22. The method of claim 15 wherein said S1 is at least 75% G and C.

23. The method of claim 15 wherein said S2 comprises a series of multiplets of nucleotides, said multiplets taken in order corresponding to at least 50% complementary multiplets at respectively ordered positions located alternately within sequences contiguous with the 3′

ends of each of said complementary single stranded polynucleotides.

24. The method of claim 23 wherein said S2 consists solely of said multiplets.

25. The method of claim 23 wherein said multiplets are quartets.

26. The method of claim 15 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

27. The method of claim 15 wherein said method is carried out at at least a 100-fold excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide sequence and its complement.

28. A method comprising repetition of the steps of claim 15 wherein extended polynucleotide primer has an inverted repeat wherein one of the sequences of the inverted repeat is identical to the polynucleotide primer.

29. A method comprising repetition of the steps (5)-(7) of claim 15 wherein the number of said copies of said polynucleotide sequence and its complement is increased by at least a factor of a thousand.

30. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, said analyte having complementary single stranded polynucleotide sequences and nucleotide sequences contiguous with the 3′

ends of said complementary single stranded polynucleotide sequences, said method comprising the steps of;

(a) providing in combination (1) said sample, (2) a polynucleotide primer comprised of at least a sequence of 16 nucleotides terminating at its 3′

end in a 4 to 8 nucleotide sequence (S1) that is complementary with the 3′

ends of both of said complementary single stranded polynucleotide sequences, wherein said polynucleotide primer has a specifically designed sequence of at least 8 nucleotides (S2) that is 5′

of said S1, said S2 being 50 to 80% complementary to each of the nucleotide sequences contiguous with the 3′

ends of said complementary single stranded polynucleotide sequences, (3) nucleoside triphosphates and (4) template dependent polynucleotide polymerase;

(b) incubating said combination under conditions for either wholly or partially sequentially or concomitantly (1) dissociating complementary sequences of said analyte into single stranded polynucleotides, (2) hybridizing said polynucleotide primer with the 3′

end of said single stranded polynucleotide sequences, (3) extending said polynucleotide primer along said single stranded polynucleotide sequences to provide a first complementary pair of extended polynucleotide primers, (4) dissociating said first complementary pair of extended polynucleotide primers from said single stranded polynucleotide sequences, (5) hybridizing single stranded extended polynucleotide primers from said first complementary pair with said polynucleotide primer, (6) extending said polynucleotide primer along said single stranded extended polynucleotide primers to provide a second complementary pair of extended polynucleotide primers, (7) dissociating said second complementary pair of extended polynucleotide primers from said single stranded extended polynucleotide primers, and (8) repeating steps (5)-(7) above,

wherein steps (a) and (b) are performed wholly or partially sequentially or concomitantly; and

(c) detecting extended polynucleotide primers, the presence thereof indicating the presence of said polynucleotide analyte.

31. The method of claim 30 wherein said S2 is at least 60% complementary with each of said nucleotide sequences contiguous with the 3′

ends of said complementary single stranded polynucleotide sequences.

32. The method of claim 30 wherein said single stranded polynucleotide sequences or said first or second extended polynucleotide primer is DNA.

33. The method of claim 30 wherein said polynucleotide primer is 24 to 90 nucleotides in length.

34. The method of claim 30 wherein said S2 contains at least one ambiguous nucleotide capable of binding to A, T, G and C.

35. The method of claim 34 wherein said ambiguous nucleotide is inosine.

36. The method of claim 30 wherein said S1 is at least 75% G and C.

37. The method of claim 30 wherein said S2 comprises a series of multiplets of nucleotides, said multiplets taken in order corresponding to at least 50% complementary multiplets at respectively ordered positions located alternately at a sequence contiguous with the 3′

ends of each of said complementary single stranded polynucleotide sequences.

38. The method of claim 37 wherein said S2 consists solely of said multiplets.

39. The method of claim 37 wherein said multiplets are quartets.

40. The method of claim 30 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

41. The method of claim 30 wherein said method is carried out at at least a 100-fold excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide analyte.

42. A method comprising repetition of steps (5)-(7) of claim 30 wherein the number of said copies of said single stranded polynucleotide sequences is increased by at least a factor of a thousand.

43. The method of claim 30 wherein said polynucleotide primer is labeled with a reporter group.

44. The method of claim 43 wherein said reporter group is selected from the group consisting of ligands, fluorescers, chemiluminescers, catalysts, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, surfaces and small organic molecules.

45. The method of claim 30 wherein said single stranded polynucleotides contain a sequence that when hybridized to its complementary sequence can be bound specifically by a receptor.

46. The method of claim 45 wherein said receptor is selected from the group consisting of repressors, activators, and restriction enzymes.

47. The method of claim 30 wherein said detection includes detection of a specific DNA sequence.

48. The method of claim 30 comprising identifying complementary single stranded polynucleotide sequences of said analyte, together with nucleotide sequences contiguous with the 3′

ends thereof, and preparing said polynucleotide primer.