US5573906A
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Detection of nucleic acids using a hairpin forming oligonucleotide primer and an energy transfer detection system
Assignee: Roche Molecular Systems Inc
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Filing: 1993-03-22
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Granted: 1996-11-12
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Expires: 2013-03-22
Abstract
Abstract A process for the detection of a nucleic acid sequence in a homogeneous assay format using an energy transfer system is disclosed. This process utilizes a 5' labeled primer containing a selfcomplementary sequence in an amplification or extension process together with a subsequent detection step using a 3' labeled probe for the amplified or extended region. The labels will be close together in space after hybridizing the probe close to the short piece of double-stranded DNA resulting from backfolding of the selfcomplementary region of the primer which has been incorporated into the amplified or extended product. A new primer for use in this process is also disclosed.
Inventors
- 👤 Wilhelm Bannwarth
- 👤 Francis Muller
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 03:50
CPC Classifications
C; C12; C12Q; C12Q1/00; C12Q1/68; C12Q1/6813; C12Q1/6816; C12Q1/6818; C07; C07H; C07H21/00; C12Q1/6844; C12Q1/6853
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
365 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected, and wherein primer extension occurs at a temperature which is high enough to prevent complete internal backfolding of Pc to Pp, utilizing a thermostable enzyme; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula 3'-Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 2
Dependent Claim
365 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected, and wherein primer extension occurs at a temperature which is high enough to prevent complete internal backfolding of Pc to Pp, utilizing a thermostable enzyme; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula 3'-Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 3
Dependent Claim
17 words
The process of claim 1, wherein X is an energy donor and Y is an energy acceptor.
Claim 4
Dependent Claim
17 words
The process of claim 1, wherein X is an energy acceptor and Y is an energy donor.
Claim 5
Dependent Claim
13 words
The process of claim 1, wherein the energy donor is a lumazine chromophore.
Claim 6
Dependent Claim
12 words
The process of claim 1, wherein the energy acceptor is a bathophenanthroline-ruthenium-II-complex.
Claim 7
Dependent Claim
30 words
The process of claim 1, wherein L consists of two propanediol units linked together by a phosphate group and attached via phosphate groups to the oligonucleotide sequences Pc and Pp.
Claim 8
Dependent Claim
14 words
The process of claim 1, wherein amplification is accomplished by the polymerase chain reaction.
Claim 9
Dependent Claim
102 words
A primer of the general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group, consisting of two propanediol units linked together by a phosphate group and attached via phosphate groups to the oligonucleotide sequences Pc and Pp provided that during the formation of said primer extension sequence during said amplification, oligonucleotide Pc is not completely internally backfolded, and X is an energy donor or acceptor.
Claim 10
Dependent Claim
102 words
A primer of the general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group, consisting of two propanediol units linked together by a phosphate group and attached via phosphate groups to the oligonucleotide sequences Pc and Pp provided that during the formation of said primer extension sequence during said amplification, oligonucleotide Pc is not completely internally backfolded, and X is an energy donor or acceptor.
Claim 11
Dependent Claim
11 words
The primer of claim 8, wherein X is an energy donor.
Claim 12
Dependent Claim
11 words
The primer of claim 8, wherein X is an energy acceptor.
Claim 13
Dependent Claim
11 words
The primer of claim 8, wherein X is a lumazine chromophore.
Claim 14
Dependent Claim
10 words
The primer of claim 8, wherein X is a bathophenanthroline-ruthenium-II-complex.
Claim 15
Dependent Claim
48 words
A kit for amplifying at least one specific nucleic acid sequence in a sample containing a nucleic add or a mixture of nucleic acids at least one of which is suspected of containing said sequence, which kit comprises a primer of formula I as defined in claim 8.
Claim 16
Dependent Claim
11 words
The kit of claim 13 which also includes a detection probe.
Claim 17
Dependent Claim
14 words
A non-nucleotidylic linker group having the formula --O--P(O).sub.2.sup.- --O--(CH.sub.2).sub.3 --O--P(O).sub.2.sup.- --O--(CH.sub.2).sub.3 --O--P(O).sub.2.sup.- --O--. 16.
Claim 18
Dependent Claim
14 words
A non-nucleotidylic linker group having the formula --O--P(O).sub.2.sup.- --O--(CH.sub.2).sub.3 --O--P(O).sub.2.sup.- --O--(CH.sub.2).sub.3 --O--P(O).sub.2.sup.- --O--. 16.
Claim 19
Dependent Claim
341 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula ' -X-Pc-L-Pp-3' wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence, and wherein primer extension occurs at a temperature which is high enough to prevent complete backfolding of Pc to Pp, utilizing a thermostable enzyme; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 20
Dependent Claim
341 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula ' -X-Pc-L-Pp-3' wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence, and wherein primer extension occurs at a temperature which is high enough to prevent complete backfolding of Pc to Pp, utilizing a thermostable enzyme; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 21
Dependent Claim
359 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected, and wherein primer extension occurs at a temperature of at least about 70° C., using a thermostable enzyme; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 22
Dependent Claim
359 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected, and wherein primer extension occurs at a temperature of at least about 70° C., using a thermostable enzyme; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 23
Dependent Claim
335 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence, and wherein the temperature at which primer extension occurs is at least about 70° C., using a thermostable enzyme; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula 3'-Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 24
Dependent Claim
335 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence, and wherein the temperature at which primer extension occurs is at least about 70° C., using a thermostable enzyme; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula 3'-Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 25
Dependent Claim
341 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 26
Dependent Claim
341 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) amplifying the nucleic acid sequence to be detected by means of a chain extension reaction utilizing a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' (I) wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, and a second oligonucleotide primer substantially complementary to a part of the other strand of the nucleic acid sequence to be detected; b) separating after a last amplification cycle the primer extension products of step (a) from their complementary sequences to produce single-stranded molecules containing a primer of formula I; c) treating said single-stranded molecules containing a primer of formula I with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or is an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to said single-stranded molecules containing a primer of formula I; and d) determining whether an energy transfer takes place as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 27
Dependent Claim
316 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 28
Dependent Claim
316 words
A process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample suspected of containing said sequence, which process comprises: a) treating the sample with a first oligonucleotide primer of general formula 5'-X-Pc-L-Pp-3' wherein Pp is an oligonucleotide sequence substantially complementary to a part of one strand of the nucleic acid sequence to be detected, Pc is an oligonucleotide sequence substantially complementary to and not longer than the sequence Pp, L is a non-nucleotidylic linker group selected so as to allow efficient backfolding between sequences Pc and Pp, and X is an energy donor or acceptor, under hybridizing conditions such that for each different nucleic acid sequence to which primer I is hybridized, an extension product of said primer is created which is complementary to said nucleic acid sequence; b) separating said extension products of said primer I from their complementary sequences to produce single-stranded molecules; c) treating said single-stranded molecules with an oligonucleotide probe of the general formula ' -Y-Pr-5' (II) wherein Y is an energy acceptor when X in the primer of formula I is an energy donor, or an energy donor when X in the primer of formula I is an energy acceptor, and Pr is an oligonucleotide sequence complementary to a part of the amplified single-stranded molecules containing the primer of formula I above and selected so as to guarantee a distance between X and Y that allows for energy transfer between X and Y after backfolding of the sequence Pc and hybridization of the sequence Pr to said single-stranded molecules such that an energy transfer can take place, under conditions allowing hybridization of the sequences Pc to Pp by backfolding and of Pr to the extension product of Primer I; and d) determining whether energy transfer occurs as a means for detecting the presence or absence of the nucleic acid sequence to be detected.
Claim 29
Dependent Claim
12 words
The process of claim 1 wherein the thermostable enzyme is Taq polymerase.
References Cited (Backward Citations)
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