Methods for reducing non-specific priming in DNA detection

• US 5,567,583 A
• Filed: 05/26/1994
• Issued: 10/22/1996
• Est. Priority Date: 12/16/1991
• Status: Expired due to Fees

• Current Assignee: Biotronics Corporation
• Original Assignee: Biotronics Corporation
• Inventors: Wang, Chang-Ning J., Wu, Kai-Yuan
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): ARTHUR, LISA BENNETT
• Application Number:
  US08/250,849
• Time in Patent Office: 880 Days
• Field of Search: 435/6, 435/91.2, 935/77, 935/78
• US Class Current: 435/6.12
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2537/1373 Displacement by a nucleic acid
• CPC Class Codes: C12Q 2537/163 blocking probe
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels

Abstract

A method for detecting a target nucleic acid, which method includes the steps of (i) amplifying the target nucleic acid to obtain an amplification product using a polymerase, a first primer with or without a segment noncontiguous to a first priming sequence, and a second primer with or without a segment noncontiguous to a second priming sequence in the presence of an oligonucleotide which is incapable of acting as a primer for the polymerase, wherein the oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of the first primer; and (ii) detecting the presence of the target nucleic acid by monitoring the amplification thereof.

Claims

1. A method for detecting a target nucleic acid, which method comprises the steps of:

amplifying the target nucleic acid to obtain an amplification product using a polymerase, a first primer with or without a segment noncontiguous to a first priming sequence, and a second primer with or without a segment noncontiguous to a second priming sequence in the presence of an oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said first primer; and

detecting the presence of the target nucleic acid by monitoring the amplification thereof.

2. The method of claim 1, wherein said detecting step is performed by monitoring said amplification product of the target nucleic acid on a gel after electrophoresis.

3. The method of claim 1, wherein a first fluorophore is covalently attached to said first primer and a second fluorophore is covalently attached to said oligonucleotide, with one of said first and second fluorophores being a donor fluorophore and the other being an acceptor fluorophore, so that when said first primer and said oligonucleotide are hybridized, said donor fluorophore and said acceptor fluorophore are in close proximity to allow resonance energy transfer therebetween;

and, further, said detecting step is performed by monitoring fluorescent emission change of said acceptor fluorophore upon irradiation of said donor fluorophore with an excitation light, said change being a function of the extent of said first primer being dissociated from said oligonucleotide and being incorporated into said amplification product of the target nucleic acid.

4. The method of claim 1, wherein said first primer includes a segment noncontiguous to said first priming sequence.

5. The method of claim 4, wherein said oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides in said noncontiguous segment of said first primer.

6. The method of claim 1, wherein said first primer does not include a segment noncontiguous to said first priming sequence.

7. The method of claim 1, wherein said amplifying step is performed in the presence of an additional oligonucleotide which is incapable of acting as a primer for said polymerase, wherein said additional oligonucleotide has at least 5 consecutive nucleotides fully complementary to at least 5 consecutive nucleotides of said second primer.

8. The method of claim 1, wherein each of said first primer, said second primer and said oligonucleotide contains 10-50 nucleotides.

9. The method of claim 8, wherein each of said first primer, said second primer and said oligonucleotide contains 15-40 nucleotides.

10. The method of claim 1, wherein said oligonucleotide has at least 8 consecutive nucleotides fully complementary to at least 8 consecutive nucleotides of said first primer.

11. The method of claim 1, wherein the concentration of said oligonucleotide is 0.3-5.0 times that of said first primer.

12. The method of claim 11, wherein the concentration of said oligonucleotide is 0.5-2.5 times that of said first primer.

13. The method of claim 12, wherein each of said first primer, said second primer and said oligonucleotide contains 10-50 nucleotides.

14. The method of claim 12, wherein said oligonucleotide has at least 8 consecutive nucleotides fully complementary to at least 8 consecutive nucleotides of said first primer.

15. The method of claim 13, wherein said oligonucleotide has at least 8 consecutive nucleotides fully complementary to at least 8 consecutive nucleotides of said first primer.

16. The method of claim 15, wherein a first fluorophore is covalently attached to said first primer and a second fluorophore is covalently attached to said oligonucleotide, with one of said first and second fluorophores being a donor fluorophore and the other being an acceptor fluorophore, so that when said first primer and said oligonucleotide are hybridized, said donor fluorophore and said acceptor fluorophore are in close proximity to allow resonance energy transfer therebetween;

and, further, said detecting step is performed by monitoring fluorescent emission change of said acceptor fluorophore upon irradiation of said donor fluorophore with an excitation light, said change being a function of the extent of said first primer being dissociated from said oligonucleotide and being incorporated into said amplification product of the target nucleic acid.