Method for preventing high molecular weight products during amplification

• US 9,447,476 B2
• Filed: 10/16/2013
• Issued: 09/20/2016
• Est. Priority Date: 10/18/2012
• Status: Active Grant

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Inventors: Babiel, Reiner, Bergmann, Frank, Sizmann, Dorothea
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US14/055,090
• Publication Number:
  US 20140113279A1
• Time in Patent Office: 1,070 Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• 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 1/707 non-A, non-B Hepatitis, excluding hepatitis D
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

The present invention is directed to improved methods for amplifying and detecting a nucleic acid target that may be present in a biological sample comprising a primer pair for generating an amplicon, wherein at least one primer is modified at the 5′ terminus with a polyN sequence being non-complementary to the target sequence, and a detectable probe specific for said amplicon or a DNA binding dye. The formation of high molecular weight products during amplification is prevented or partly or completely suppressed. The invention further provides reaction mixtures and kits comprising said primers for preventing or suppressing the formation of high molecular weight products during amplification and detection of the nucleic acid target.

Claims

1. A method for amplifying and detecting a target nucleic acid of HCV in a biological sample suspected of containing the target nucleic acid, said method comprising:

a) contacting nucleic acids in said biological sample with amplification reagents comprising at least a DNA polymerase, nucleoside triphosphate monomers, at least one extended forward primer specific for said target nucleic acid, or at least one extended reverse primer specific for said target nucleic acid, or a combination thereof, for generating at least one amplicon, and at least one detectable probe specific for said amplicon, or at least one DNA binding dye, wherein said at least one extended forward primer or said at least one reverse primer comprises a polyN sequence of between 2 and 10 nucleotides in length added to the 5′

terminus of the primer, said polyN sequence being non-complementary to the target sequence and comprising 100% of either (i) one defined selected nucleotide or (ii) two defined selected nucleotides, wherein said primer is sufficiently complementary to hybridize with said target nucleic acid,

b) incubating said nucleic acids with said amplification reagents for a period of time and under conditions sufficient for an amplification reaction to occur, wherein the formation of high molecular weight products during amplification is reduced as compared to amplification without a primer comprising a polyN sequence; and

c) detecting said amplicon via said at least one detectable probe or said at least one DNA binding dye.

2. The method according to claim 1, wherein said polyN sequence comprises a polyA, a polyT, a polyAT, a polyU, a polyC or a polyG sequence.

3. The method according to claim 1, wherein said polyN sequence is between 4 to 6 nucleotides in length.

4. The method according to claim 1, wherein said amplification reaction terminates after at least 50 cycles.

5. The method according to claim 1, wherein at least step b) is performed in the presence of an enzyme comprising uracil-DNA glycosylase activity.

6. The method according to claim 1, wherein said at least one extended forward primer comprises a sequence from the group consisting of SEQ ID NO:

3, SEQ ID NO;

5, SEQ ID NO;

7, SEQ ID NO;

9, SEQ ID NO;

11, SEQ ID NO;

13, SEQ ID NO;

15, SEQ ID NO;

19, SEQ ID NO;

21, and SEQ ID NO;

23.

7. The method according to claim 1, wherein said at least one detectable probe comprises a sequence from the group consisting of SEQ ID NO:

27, SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

30 and the complementary sequences thereof.

8. The method according to claim 1, wherein said at least one extended reverse primer comprises a sequence from the group consisting of SEQ ID NO:

4, SEQ ID NO;

6, SEQ ID NO;

8, SEQ ID NO;

10, SEQ ID NO;

12, SEQ ID NO;

14, SEQ ID NO;

16, SEQ ID NO;

20, and SEQ ID NO;

22.

9. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

4, and wherein said at least one extended forward primer comprises SEQ ID NO;

3.

10. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

6, and wherein said at least one extended forward primer comprises SEQ ID NO;

5.

11. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

8, and wherein said at least one extended forwarded primer comprises SEQ ID NO;

7.

12. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

10, and wherein said at least one extended forwarded primer comprises SEQ ID NO;

9.

13. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

14, and wherein said at least one extended forwarded primer comprises SEQ ID NO;

13.

14. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

16, and wherein said at least one extended forwarded primer comprises SEQ ID NO;

15.

15. The method according to claim 1, wherein said at least one detectable probe comprises SEQ ID NO:

29 or the complementary sequence thereof.

16. The method according to claim 3, wherein said polyN sequence is between 4 to 6 consecutive adenine (A) nucleotides in length.

17. The method according to claim 1, wherein said at least one extended reverse primer comprises SEQ ID NO:

12, and wherein said at least one extended forward primer comprises SEQ ID NO;

11.