Method for introducing defined sequences at the 3'end of polynucleotides

• US 5,679,512 A
• Filed: 10/20/1993
• Issued: 10/21/1997
• Est. Priority Date: 07/31/1992
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behringwerke Aktiengesellschaft (Sanofi )
• Inventors: Chen, Yan, Ullman, Edwin F., Hahnenberger, Karen M., Laney, Maureen
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): CAMPBELL, EGGERTON A
• Application Number:
  US08/140,349
• Time in Patent Office: 1,462 Days
• Field of Search: 435/91.1, 435/6, 935/78
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/163 blocking probe

Abstract

A method is disclosed for extending a primer to produce a single stranded polydeoxynucleotide that has two or more defined sequences. A combination is provided which comprises a template polynucleotide, a blocker polynucleotide, a primer polynucleotide and a polynucleotide Q. The template polynucleotide has three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3'"'"' of T3 and wherein the 5'"'"' end of T3 is 5'"'"' of the 5'"'"' end of T2. The primer polynucleotide has a second defined sequence at its 3'"'"' end that is hybridizable with T1. The blocker polynucleotide has sequence B1 that is hybridizable with T3. Polynucleotide Q has sequences S1 and S2 wherein S1 is 3'"'"' of S2 and homologous with T2 and S2 is complementary to a first defined sequence that is to be introduced at the 3'"'"' end of the polynucleotide primer, when it is extended during the method of the invention. Polynucleotide Q is either attached to the 5'"'"' end of the blocker polynucleotide or present as a separate reagent. The primer is extended along the template polynucleotide and along at least a portion of sequence T2 and thereafter along the polynucleotide Q to give a single stranded polynucleotide having two or more defined sequences.

Claims

1. A method for producing a single stranded polynucleotide having two or more defined nucleic acid sequences, said method comprising the steps of:

(a) hybridizing a sequence B1 of a blocker polynucleotide to a complementary sequence T3 within a template polynucleotide ("template"), said template being comprised of a sequence T1 and a sequence T2, wherein the 5'"'"' end of said T3 is 5'"'"' of the 5'"'"' end of said T2 and said T2 and T3 are 5'"'"' to and non-contiguous with said sequence T1,

(b) hybridizing the 3'"'"' end of a polynucleotide primer comprising a second defined nucleic acid sequence to said T1 wherein step (b) is performed prior to, after, or simultaneously with step (a), and

(c) extending said primer, by means of a polymermase, along said template and along at least a portion of said T2 and thereafter along a polynucleotide Q having sequences S1 and S2 wherein S1 is 3'"'"' of S2 and substantially identical with said T2 to give an extended primer having a first defined nucleic acid sequence at its 3'"'"' end that is complementary to S2, wherein polynucleotide Q is either attached to the 5'"'"' end of said blocker polynucleotide or is present as a separate reagent, wherein said polymerase has little or no 5'"'"'-3'"'"' exonuclease activity during said extending, and wherein the 3'"'"'-end of said polynucleotide Q, when present as a separate reagent, is not extended by said polymerase during said extending.

2. A method for producing from a primer polynucleotide a single stranded polydeoxynucleotide having two segments that are non-contiguous and complementary with each other, said method comprising the steps of:

(a) providing in combination (a) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3'"'"' of said T2 and T3 and the 3'"'"' end of said T3 is oontiguous with or lies within said T2, (b) a pdmer polynucleotide whose 3'"'"' end is hybridizable with said T1 (c) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, and (d) a polynucleotide Q having sequence S1 and sequence S2 wherein said Q is either attached to the 5'"'"' end of said blocker polynucleotide or present as a separate reagent and wherein S1 is 3'"'"' of said S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3'"'"' end of said pdmer polynucleotide, and

(b) subjecting said combination to conditions for extending said primer, by means of a polymerase, along said template polynucleotide and along at least a portion of said T2, and thereafter along said polynucleotide Q, wherein said polymerase has little or no 5'"'"'-3'"'"' exonuclease activity under said conditions for extending, and wherein the 3'"'"'-end of said polynucleotide Q, when present as a separate reagent, is not extended during said extending.

3. The method of claim 2 wherein at least a five base sequence within said B1 is comprised of at least 80% G and C nucleotides.

4. The method of claim 2 wherein said S1 is from 5 to 50 nucleotides in length.

5. The method of claim 2 wherein a sequence comprising the 5'"'"' end of said S1 is complementary to a sequence comprising the 5'"'"' end of said B1.

6. The method of claim 2 wherein the 5'"'"' end of said S1 is not complementary to the 5'"'"' end of said B1.

7. The method of claim 2 wherein said T2 is contiguous with said T3.

8. The method of claim 2 wherein the 3'"'"'-end of said polynucleotide Q is attached to the 5'"'"' end of said blocker polynucleotide.

9. A method of producing at least one copy of a target polynuoleotide sequence, said method comprising the step of:

providing in combination (1) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3'"'"' of said T2 and T3 and wherein the 3'"'"' end of said T3 is contiguous with or lies within said T2 and wherein said target polynucleotide sequence is located between said T1 and T3, (2) a primer polynucleotide whose 3'"'"' end is hybridizable with said T1, (3) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (4) a polynucleotide Q having sequences S1 and S2 wherein said Q is either attached to the 5'"'"' end of said blocker polynucleotide or present as a separate reagent and wherein S1 is 3'"'"' of S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3'"'"' end of said primer polynucleotide, (5) DNA polymerase and (6) deoxynucleoside triphosphates under conditions wherein;

(A) said blocker becomes hybridized to said template polynucleotide, (B) said primer becomes hybridized with and extended along said target polynucleotide sequence of said template polynucleotide and along at least a portion of said T2, and thereafter along said polynucleotide Q to form a duplex, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer containing a copy of said target sequence, wherein the 3'"'"'-end of said polynucleotide Q, when present as a separate reageent, is not extended by said DNA polymerase.

10. The method of claim 9 which comprises the step of examining for the presence of said extended primer.

11. The method of claim 9 wherein at least a five base sequence of said B1 is comprised of at least 80% G and C nucleotides.

12. The method of claim 9 wherein said S1 is from 5 to 50 nucleotides in length.

13. The method of claim 9 wherein a sequence comprising the 5'"'"' end of said S1 is complementary to a sequence comprising the 5'"'"' end of said B1.

14. The method of claim 9 wherein the 5'"'"' end of said S1 is not complementary to the 5'"'"' end of said B1.

15. The method of claim 9 wherein said T2 is contiguous with said T3.

16. The method of claim 9 wherein steps (C) and (D) are repeated.

17. The method of claim 9 wherein the 3'"'"'-end of said polynucleotide Q is attached to the 5'"'"' end of said blocker polynucleotide.

18. A method of forming multiple copies of a target polynucleotide sequence, said method comprising the steps of:

(a) providing in combination (1) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-oontiguous and 3'"'"' of said T2 and T3 and wherein the 3'"'"' end of said T3 is contiguous with or lies within said T2 and wherein said target polynucleotide sequence is located between said T1 and T3, (2) a primer polynucleotide whose 3'"'"' end is hybridizable with said T1, (3) a blocker polynucleotlde with sequence B1, said B1 being hybridizable with said T3, (4) a poly-nucleotide Q having sequences S1 and S2 wherein said Q is either attached to the 5'"'"' end of said blocker polynucleotide or present as a separate reagent and wherein S1 is 3'"'"' of S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3'"'"' end of said primer polynucleotide, (5) DNA polymerase and (6) deoxynucleoside triphosphates under conditions wherein;

(A) said blocker becomes hybridized to said template polynucleotide, (B) said primer becomes hybridized with and extended along said target polynucleotide sequence of said template polynucleotide and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer containing a copy of said target sequence, wherein the 3'"'"'-end of said polynuoleotide Q, when present as a separate reagent, is not extended by said DNA polymerase and

(b) repeating steps (C) and (D).

19. The method of claim 18 which comprises examining for the presence of said extended primer.

20. The method of claim 18 wherein at least a five base sequence said B1 is comprised of at least 80% G and C nucleotides.

21. The method of claim 18 wherein said S1 is from 5 to 50 nucleotides in length.

22. The method of claim 18 wherein a sequence comprising the 5'"'"' end of said S1 is complementary to a sequence comprising the 5'"'"' end of said B1.

23. The method of claim 18 wherein the 5'"'"' end of S1 is not complementary to the 5'"'"' end of said B1.

24. The method of claim 18 wherein said T2 is contiguous with said T3.

25. The method of claim 18 wherein the 3'"'"'-end of said polynucleotide Q is attached to the 5'"'"' end of said blocker polynucleotide.

26. A method of forming multiple copies of a target polynucleotide sequence, said method comprising the steps of:

(a) providing in combination (1) a template polynucleotide having three sequences T1, T2 and T3 wherein T1 is non-contiguous and 3'"'"' of said T2 and T3 and wherein the 5'"'"' end of said T3 is 5'"'"' of the 5'"'"' end of said T2 and wherein said target polynucleotide sequenoe is located between said T1 and T3, (2) a primer polynucleotide whose 3'"'"' end is hybridizable with said T1, (3) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (4) a polynucleotide Q having sequences S1 and S2 wherein the 3'"'"'-end of said Q is attached to the 5'"'"' end of said blocker polynucleotide and wherein S1 is 3'"'"' of S2 and substantially identical to said T2 and wherein said S2 is a sequence that is substantially identical to at least the 3'"'"' end of said primer polynucleotide, (5) DNA polymerase and (6) deoxynudeoside triphosphates under conditions wherein;

(A) said blocker becomes hybridized to said template polynucleotide, (B) said primer becomes hybridized with and extended along said target polynucleotide sequence of said template polynucleotide and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, (C) said extended primer is dissociated from the said duplex, and (D) said primer hybridizes with and is extended along said extended primer to form a duplex comprising extended primer containing a copy of said target sequence, and

(b) repeating steps (C) and (D).

27. The method of claim 26 which comprises examining for the presence of said extended primer.

28. The method of claim 26 wherein at least a five base sequence said B1 is comprised of at least 80% G and C nucleotides.

29. The method of claim 26 wherein said S1 is from 5 to 50 nucleotides in length.

30. The method of claim 26 wherein the 5'"'"' end of said S1 is complementary to the 5'"'"' end of said B1.

31. The method of claim 26 wherein the 5'"'"' end of said S1 is not complementary to the 5'"'"' end of said B1.

32. The method of claim 26 wherein said T2 is contiguous with said T3.

33. The method of claim 26 wherein said T2 is not contigous with said T3.

34. A kit comprising in packaged combination:

(a) a primer polynucleotide having a sequence at its 3'"'"' end hybridizable with a first sequence T1 in a target polynucleotide

(b) a blocker polynucleotide having a sequence B1, said B1 being hybridizable with a sequence T3 in said target polynucleotide wherein said T3 is 3'"'"' of said T1 and the 5'"'"' end of said T3 is 5'"'"' of the 5'"'"' end of a sequence T2, and

(c) a polynucleotide Q having sequences S1 and S2 wherein S1 is 3'"'"' of said S2 and substantially identical to said T2 in said template polynucleotide and wherein said S2 is a sequence that is substantially identical to at least the 3'"'"' end portion of said primer.

35. The kit of claim 34 wherein said polynucleotide Q is a part of said blocker polynucleotide.

36. The kit of claim 34 wherein said polynucleotide Q is separate from said blocker polynucleotide.

37. The kit of claim 34 which comprises template dependent DNA polymerase.

38. The kit of claim 34 which comprises deoxynucleoside triphosphates.

39. The kit of claim 34 wherein said primer is labeled with a reporter molecule.