Method for detecting a nucleic acid sequence
Assignee3Cr Bioscience Ltd
InventorsJohn Holme, Nisha Jain, Steven Asquith
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Abstract
The present invention relates to a method for detecting one or more target sequences in a sample by amplification. Kits and compositions for use with the method are also provided.
Claims64
A method for detecting one or more target sequences in a sample by amplification, the method comprising:
- a) providing an aqueous composition comprising:
- i) one or more forward oligonucleotide primers having a 5′ region which is a tag sequence and a 3′ region specific for a target sequence;
- ii) a reverse oligonucleotide primer, such that a forward and the reverse primer operably form a primer pair;
- iii) one or more probes having a reporter label and a quencher label, each probe comprising a sequence having at least 50% identity to the tag sequence of a forward oligonucleotide primer;
- and
- iv) one or more enhancer oligonucleotide primers comprising a sequence having at least 50% identity to the sequence of a probe;
- b) incubating the primers and probes with the sample and at least a polymerase;
- and
- c) performing polymerase chain reaction (PCR) on the sample to generate tagged nucleic acids, whereby the probe(s) can bind the tagged nucleic acids.
A kit for use in a nucleic acid amplification process, comprising:
- i) one or more forward oligonucleotide primers having a 5′ region which is a tag sequence and a 3′ region specific for a target sequence;
- ii) a reverse oligonucleotide primer, such that a forward and the reverse primer operably form a primer pair;
- iii) one or more probes having a reporter label and a quencher label, each probe comprising a sequence having at least 50% identity to the tag sequence of a forward oligonucleotide primer;
- iv) one or more enhancer oligonucleotide primers comprising a sequence having at least 50% identity to the sequence of a probe;
- and
- v) a polymerase.
The method or kit according to any one of the preceding claims, wherein the polymerase is a DNA polymerase.
The method or kit according to any one of the preceding claims, wherein the polymerase is a fragment or domain or a derivative of Taq polymerase.
The method or kit according to any one of the preceding claims, wherein the polymerase is a modified polymerase.
The method or kit according to any one of the preceding claims, wherein the one or more enhancer oligonucleotide primers comprise at least two distinct regions, each region comprising a sequence having at least 50% identity to that of the one or more probes.
The method or kit according to any one of the preceding claims, wherein the one or more probes comprise a sequence having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to the tag sequence of a forward oligonucleotide primer.
The method or kit according to any one of the preceding claims, wherein the one or more probes comprise a sequence identical to the tag sequence of a forward oligonucleotide primer.
The method or kit according to any one of the preceding claims, wherein the one or more enhancer oligonucleotide primers comprise a sequence having at least 60%, 70%, 80%, 90%, 95%, 98% or 99% identity to the sequence of a probe.
The method or kit according to any one of the preceding claims, wherein the one or more enhancer oligonucleotide primers comprise a sequence identical to the sequence of a probe.
The method according to any one of the preceding claims, wherein at least 4 cycles of PCR are performed.
The method according to any one of the preceding claims, further comprising:
- d) measuring the signal generated by the binding of the probes to the tagged nucleic acids.
The method or kit according to any one of the preceding claims, wherein the composition or kit further comprises a quaternary ammonium salt.
A (modified) polymerase with reduced or no exonuclease function or activity (herein abbreviated to P-ENF) for use in a nucleic acid amplification method or process.
A method of conducting nucleic acid amplification using a P-ENF (such as according to any preceding claims).
An aqueous composition suitable for nucleic acid amplification comprising:
- a) a buffer;
- b) dNTPs;
- c) a P-ENF;
- d) optionally, a forward primer and a reverse primer;
- and
- e) a source of divalent cations.
The use of a P-ENF in a nucleic acid amplification method.
The use of a quaternary ammonium salt (QAS) in a nucleic acid amplification method.
A process for conducting nucleic acid amplification using quaternary ammonium salt.
An aqueous composition for use in a nucleic acid amplification process, the composition comprising:
- a) a buffer;
- b) dNTPs;
- c) a DNA polymerase (Such as a P-ENF);
- d) optionally, a forward primer and a reverse primer;
- e) a quaternary ammonium salt (QAS);
- and
- f) a source of divalent cations.
Dependencies are inferred from the claim text.