US5348853A — Method for reducing non-specific priming in DNA a…

Claim 1

Independent Claim 137 words

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.

Claim 2

Dependent Claim 137 words

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.

Claim 3

Dependent Claim 24 words

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

Claim 4

Dependent Claim 29 words

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.

Claim 5

Dependent Claim 68 words

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.

Claim 6

Dependent Claim 92 words

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.

Claim 7

Independent Claim 319 words

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.

Claim 8

Independent Claim 319 words

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.

Claim 9

Dependent Claim 25 words

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.

Claim 10

Dependent Claim 12 words

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

Claim 11

Dependent Claim 81 words

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.

Claim 12

Dependent Claim 31 words

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.

Claim 13

Dependent Claim 79 words

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.

Claim 14

Dependent Claim 96 words

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.

Claim 15

Dependent Claim 96 words

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.

Claim 16

Dependent Claim 46 words

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.

Claim 17

Dependent Claim 211 words

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.

Claim 18

Dependent Claim 211 words

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.

Claim 19

Dependent Claim 45 words

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.

Claim 20

Dependent Claim 52 words

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.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1997-09-19	1998-03-21	1998-09-20	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2001-09-19	2002-03-21	2002-09-20	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2005-09-19	2006-03-21	2006-09-20	$7700.00	Window Closed / Lapsed

Method for reducing non-specific priming in DNA amplification

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