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

• US 6,030,774 A
• Filed: 06/07/1995
• Issued: 02/29/2000
• Est. Priority Date: 07/31/1992
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behring Diagnostics GmbH
• Inventors: Chen, Yan, Laney, Maureen, Ullman, Edwin F., Hahnenberger, Karen M.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/479,745
• Time in Patent Office: 1,728 Days
• Field of Search: 435/6, 435/91.2, 435/91.1
• 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 detecting the presence of a polynucleotide analyte, said analyte comprising a template sequence having three sequences T1, T2 and T3 wherein T1 is non-contiguous with and 3'"'"' of said T2 and T3 and the 5'"'"' end of said T3 is 5'"'"' of the 5'"'"' end of said T2, in a medium suspected of containing said analyte said method comprising:

(a) combining said medium with (1) a primer polynucleotide whose 3'"'"' end is hybridizable with said T1, (2) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (3) a polynucleotide Q having sequences S1 and S2 wherein Q is attached at its 3'"'"'-end to the 5'"'"' end of said blocker polynucleotide or is present as a separate reagent and wherein S1 is 3'"'"' of S2 and is substantially identical to said T2 and wherein said S2 is substantially identical to at least the 3'"'"' end of said primer polynucleotide, (4) DNA polymerase and (5) deoxynucleoside triphosphates under conditions wherein;

(A) said blocker becomes hybridized to said template, (B) said primer becomes hybridized with and extended along, by means of a polymerase, said template and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, 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 by said polymerase, (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 and steps (C) and (D) are repeated, and

(b) examining for the presence of said extended primer.

2. The method of claim 1 wherein at least a five base sequence within the 15 bases at the 3'"'"' end of said T3 is comprised of at least 80% G and C nucleotides.

3. The method of claim 1 wherein said S1 is from 5 to 50 nucleotides in length.

4. The method of claim 1 wherein the 5'"'"' end of said S1 is complementary to the 5'"'"' end of said B1.

5. The method of claim 1 wherein the 5'"'"' end of said S1 not complementary to the 5'"'"' end of said B1.

6. The method of claim 1 wherein said T2 homologous is contigous with said T3.

7. The method of claim 1 wherein said T2 is not contiguous with said T3.

8. The method of claim 1 wherein said polynucleotide analyte is DNA.

9. The method of claim 1 wherein said polynucleotide is RNA and said medium includes reverse transcriptase.

10. The method of claim 1 wherein said polydeoxynucleotide primer is labeled with a reporter molecule.

11. The method of claim 1 wherein said polynucleotide Q is attached to the 5'"'"' end of said blocker polynucleotide.

12. A method for detecting the presence of a polynucleotide analyte comprising a template sequence having three sequences T1, T2 and T3 wherein T1 is non-contiguous with and 3'"'"' of said T2 and T3 and the 3'"'"' end of said T3 is contiguous with or lies within said T2, in a medium suspected of containing said analyte, said method comprising:

(a) combining said medium with (1) a primer polynucleotide whose 3'"'"' end is hybridizable with said T1, (2) a blocker polynucleotide with sequence B1, said B1 being hybridizable with said T3, (3) a polynucleotide Q having sequences S1 and S2 wherein Q is attached at its 3'"'"'-end to the 5'"'"' end of said blocker polynucleotide and wherein S1 is 3'"'"' of said S2 and is substantially identical to said T2 and wherein said S2 is substantially identical to at least the 3'"'"' end of said primer polynucleotide, (4) DNA polymerase and (5) deoxynucleoside triphosphates under conditions wherein;

(A) said blocker becomes hybridized to said template, (B) said primer becomes hybridized with and extended along, by means of a polymerase, said template polynucleotide and along at least a portion of said T2 and thereafter along said polynucleotide Q to form a duplex, wherein said polymerase has little or no 5'"'"'-3'"'"' exonuclease activity under said conditions for extending, (C) the 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 and steps (C) and (D) are repeated, and

(b) examining for the presence of said extended primer.

13. The method of claim 12 wherein at least a five base sequence within the 15 bases at the 3'"'"' end of said T3 is comprised of at least 80% G and C nucleotides.

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

15. The method of claim 12 wherein the 5'"'"' end of said S1 is complementary to the 5'"'"' end of said B1.

16. The method of claim 12 wherein the 5'"'"' end of said S1 not complementary to the 5'"'"' end of said B1.

17. The method of claim 12 wherein said T2 is contigous with said T3.

18. The method of claim 12 wherein said polynucleotide analyte is DNA.

19. The method of claim 12 wherein said polynucleotide is RNA and said medium includes reverse transcriptase.

20. The method of claim 12 wherein said polydeoxynucleotide primer is labeled with a reporter molecule.