Detection of nucleic acids using a hairpin forming oligonucleotide primer and an energy transfer detection system
First Claim
1. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">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.
3 Assignments
0 Petitions
Accused Products
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.
239 Citations
21 Claims
-
1. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 21)
-
- 8. A primer of the general formula
- space="preserve" listing-type="equation">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. - View Dependent Claims (9, 10, 11, 12, 13, 14)
- 15. A non-nucleotidylic linker group having the formula
- space="preserve" listing-type="equation">--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.
-
16. 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
space="preserve" listing-type="equation">'"'"' -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
space="preserve" listing-type="equation">'"'"' -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.
-
-
17. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">'"'"' -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.
-
-
18. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">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.
-
-
19. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">'"'"' -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.
-
-
20. 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
space="preserve" listing-type="equation">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
space="preserve" listing-type="equation">'"'"' -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.
-
Specification