Method for reducing non-specific priming in DNA amplification

• US 5,348,853 A
• Filed: 12/16/1991
• Issued: 09/20/1994
• Est. Priority Date: 12/16/1991
• Status: Expired due to Fees

• Current Assignee: Biotronics Corporation
• Original Assignee: Biotronics Corporation
• Inventors: Wu, Kai-Wuan, Wang, Chang-Ning J.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Schreiber, David
• Application Number:
  US07/808,463
• Time in Patent Office: 1,009 Days
• Field of Search: 435/6, 435/91, 435/91.2
• 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 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

This invention relates to a homogeneous process for amplifying a target sequence in a nucleic acid sample and detecting amplification in the absence of a separation step. The invention further provides a method for nucleic acid amplification under conditions which substantially reduce the occurrence of nonspecific amplification. Products and an apparatus related to the homogeneous process are also described.

Claims

1. In a method for a polymerase dependent extension reaction for synthesizing an extension product complementary to a target nucleic acid within a larger nucleic acid template comprising:

combining a pair of primers with said nucleic acid template;

and performing said polymerase dependent extension reaction under conditions sufficient to synthesize said extension product, the improvement comprising;

combining said pair of primers with the nucleic acid template in the presence of at least one energy sink oligonucleotide that is incapable of acting as a primer in an extension reaction, wherein each of said at least one energy sink oligonucleotides has sufficient complementarity to at least one of said primers and is present in sufficient concentration so as to competitively inhibit binding of said primer to a non-target nucleic acid via hybridization of said energy sink oligonucleotide to said primer.

2. The method of claim 1, wherein the 3'"'"' end of at least one of said primers protrudes when hybridized to said energy sink oligonucleotide.

3. The method of claim 1, wherein the energy sink oligonucleotide has at least 10 consecutive bases perfectly complementary to a 10 base sequence in the one of the primers.

4. The method of claim 1, wherein a lagging probe having a reporting group attached thereto is hybridized to the nucleic acid template at a position downstream of the direction of elongation, the method further comprising:

utilizing elongation of the primer to displace the lagging probe from the template and

measuring a change in a signal resulting from the displacement of the lagging probe from the nucleic acid template.

5. The method of claim 1, wherein a lagging probe is hybridized to the nucleic acid template at a position downstream of the direction of elongation, and wherein the primer and the lagging probe are labeled such that the generation of a signal depends upon the juxtaposition of the labelled primer and the labelled lagging probe, the method further comprising:

utilizing elongation of the primer to displace the lagging probe from the template and

measuring a change in a signal resulting from the displacement of the lagging probe from the nucleic acid template.

6. A method for amplifying and detecting a target nucleic acid sequence in a nucleic acid template in the same vessel, the method comprising:

a) providing in the presence of the nucleic acid template, a first duplex and a second duplex, the first duplex including a first primer and a first energy sink oligonucleotide, the second duplex including a second primer and a second energy sink oligonucleotide, wherein the first energy sink oligonucleotide is of sufficient complementarity to the first primer so as to competitively inhibit binding of the first primer to a non-target sequence in the nucleic acid template and wherein the second energy sink oligonucleotide is of sufficient complementarity to the second primer so as to competitively inhibit binding of the second primer to the non-target sequence, wherein the first energy sink oligonucleotide and the second energy sink oligonucleotide are incapable of acting as extension primers in an extension reaction;

b) allowing the first primer, the first energy sink oligonucleotide, the second primer and the second energy sink oligonucleotide to hybridize to the template so that the first energy sink oligonucleotide hybridizes to the template at a position downstream of the direction of elongation of the second primer and the second energy sink oligonucleotide hybridizes to the template at a position downstream of the direction of elongation of the first primer, wherein at least one of the first energy sink oligonucleotide and the second energy sink oligonucleotide has a reporting group attached thereto;

c) subjecting the template having the primers and the energy sink oligonucleotides hybridized thereto to conditions sufficient to permit the first primer to elongate sufficiently to displace the second energy sink oligonucleotide and the second primer to elongate sufficiently to displace the first energy sink oligonucleotide from the template, and

d) measuring a change in a signal resulting from the displacement of the energy sink oligonucleotide having the reporting group attached thereto from the template.

7. The method of claim 6, wherein the reporting group is selected from the group consisting of a fluorophore, a chromophore and a specific binding agent.

8. The method of claim 7, wherein the reporting group is a fluorophore.

9. The method of claim 6, wherein at least one of the primers has a reporting group attached thereto, wherein the primer having the reporting group attached thereto and the energy sink oligonucleotide having the reporting group attached thereto are labelled such that a first signal is measurable when the labeled primer and the labeled energy sink oligonucleotide are juxtaposed and a second signal is measurable when the labeled primer and the labeled energy sink oligonucleotide are not juxtaposed with one another.

10. The method of claim 9, wherein the reporting group attached to the primer and the reporting group attached to the energy sink oligonucleotide are fluorophores with overlapping emission and excitation wavelengths.

11. The method of claim 6, wherein at least one of the primers has a reporting group attached thereto, wherein the primer having the reporting group attached thereto and the energy sink oligonucleotide having the reporting group attached thereto are labelled such that juxtaposition of the labeled primer and the labeled energy sink oligonucleotide results in quenching of a signal emitted by the reporting group attached to the primer or by the reporting group attached to the energy sink oligonucleotide.

12. A kit for use in a polymerase dependent extension reaction that utilizes a pair of primers to synthesize an extension product complementary to a target sequence in a nucleic acid template wherein binding of at least one of the primers to a non-target sequence is prevented, the kit consisting essentially of:

a duplex containing a primer and an energy sink oligonucleotide that is incapable of acting as an extension primer in an extension reaction, wherein the energy sink oligonucleotide is sufficiently complementary to the primer to competitively inhibit binding of the primer to the non-target sequence.

13. The kit of claim 12, wherein the energy sink oligonucleotide has a 5'"'"' end, and wherein the 3'"'"' end of the extension primer protrudes beyond the 5'"'"' end of the energy sink oligonucleotide when the primer and the energy sink oligonucleotide are hybridized to one another.

14. A kit for use in a polymerase dependent extension reaction that utilizes a pair of primers to synthesize an extension product complementary to a target sequence in a nucleic acid template wherein binding of at least one of the primers to a non-target sequence is prevented, the kit consisting essentially of:

a first duplex containing a first primer and a first energy sink oligonucleotide that is incapable of acting as an extension primer in an extension reaction, wherein the first energy sink oligonucleotide is complementary to the first primer; and

a second duplex containing a second primer and a second energy sink oligonucleotide that is incapable of acting as an extension primer in an extension reaction, wherein the second energy sink oligonucleotide is complementary to the second primer;

wherein the first energy sink oligonucleotide and the second energy sink oligonucleotide competitively inhibit binding of the primers to the non-target sequence, and further, wherein the first primer and the second energy sink oligonucleotide each are labeled with a different reporting group such that a first signal is measurable when the first primer and second energy sink oligonucleotide are juxtaposed and wherein a second signal is measurable when the first primer and the second energy sink oligonucleotide are not juxtaposed with one another.

15. The method of claim 14, wherein juxtaposition of the labeled primer and the labeled energy sink oligonucleotide results in quenching of a signal emitted by the reporting group attached to the first primer or by the reporting group attached to the second energy sink oligonucleotide.

16. The kit of claim 14, wherein both of the energy sink oligonucleotides have a 5'"'"' end and wherein the 3'"'"' end of at least one of the primers protrudes beyond the 5'"'"' end of its complementary energy sink oligonucleotide when the primer and the energy sink oligonucleotide are hybridized to one another.