Methods of using FET labeled oligonucleotides that include a 3'-5' exonuclease resistant quencher domain and compositions for practicing the same
OwnerQuin Chou, Dragan Spasicinventors
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Abstract
Methods and compositions are provided for detecting a primer extension product in a reaction mixture. In the subject methods, a primer extension reaction is conducted in the presence of a polymerase having 3′→5′ exonuclease activity and at least one FET labeled oligonucleotide probe that includes a 3′→5′ exonuclease resistant quencher domain. Also provided are systems and kits for practicing the subject methods. The subject invention finds use in a variety of different applications, and are particularly suited for use in high fidelity PCR based reactions, including SNP detection applications, allelic variation detection applications, and the like.
Claims53
A method for detecting the production of a primer extension product in a primer extension reaction mixture, said method comprising:
- (a) producing a primer extension mixture that includes a nucleic acid polymerase, a FET labeled oligonucleotide that includes a 3′→5′ exonuclease resistant quencher domain, and at least one of:
- (i) a nucleic acid intercalator, and
- (ii) a minor groove binder;
- (b) subjecting said primer extension mixture to primer extension reaction conditions;
- (c) detecting a change in a fluorescent signal from said FET labeled oligonucleotide probe to obtain an assay result;
- and
- (d) employing said assay result to determine whether a primer extension product is present in said mixture.
A FET labeled probe comprising nucleic acid intercalator bonded to a FET labeled oligonucleotide.
A method of monitoring of a PCR amplification reaction, said method comprising:
- (a) preparing a PCR amplification reaction mixture by combining:
- (i) a template nucleic acid;
- (ii) forward and reverse nucleic acid primers;
- (iii) deoxyribonucleotides;
- (iv) a nucleic acid polymerase;
- (v) a FET labeled oligonucleotide that includes:
- a 3′→5′ exonuclease resistant quencher domain comprising a dark quencher, a fluorescent reporter, domain comprising a fluorophore and a PCR product complementary domain;
- and
- (vi) (vi) at least one of:
- (A) a nucleic acid intercalator, and
- (B) a minor groove binder;
- (b) subjecting said PCR amplification reaction mixture to PCR amplification conditions;
- (c) monitoring said reaction mixture for a fluorescent signal from said FET labeled oligonucleotide probe to obtain an assay result;
- and
- (d) employing said assay result to monitor said PCR amplification reaction.
A method for screening a nucleic acid sample for the presence of first and second nucleic acids that differ from each other by a single nucleotide, said method comprising:
- (a) producing a primer extension mixture that includes:
- (i) said nucleic acid sample;
- (ii) a nucleic acid polymerase;
- (iii) first and second FET labeled oligonucleotide probes that are complementary to said first and second nucleic acids, respectively, wherein each of said first and second FET labeled oligonucleotides includes a 3′→5′ exonuclease resistant quencher domain;
- and:
- (iv) at least one of:
- (A) a nucleic acid intercalator, and
- (B) a minor groove binder
- (b) subjecting said primer extension mixture to primer extension reaction conditions;
- (c) detecting a change in a fluorescent signal, if any, from said first and second FET labeled oligonucleotide probes to obtain an assay result;
- and
- (d) employing said assay result to determine the presence or absence of said first and second nucleic acids in said sample.
A system for use in detecting the production of a primer extension product in a primer extension reaction mixture, said system comprising:
- (a) a FET labeled oligonucleotide that includes a 3′→5′ exonuclease resistant quencher domain;
- (b) a nucleic acid polymerase;
- and
- (c) at least one of:
- (i) a nucleic acid intercalator;
- and
- (ii) a minor groove binder.
A kit for use in detecting the production of a primer extension product in a primer extension reaction mixture, said kit comprising:
- (a) a FET labeled oligonucleotide that includes a 3′→5′ exonuclease resistant quencher domain;
- (b) at least one of:
- (i) a nucleic acid intercalator;
- and
- (ii) a minor groove binder.
In a method comprising employing fluorescently labeled nucleic acid detector and a polymerase, the improvement comprising:
- employing a FET labeled oligonucleotide that includes a 3′→5′ exonuclease resistant quencher domain as said fluorescently labeled nucleic acid detector, and at least one of:
- (a) a nucleic acid intercalator;
- and
- (b) a minor groove binder.
Dependencies are inferred from the claim text.