Methods of using FET labeled oligonucleotides that include a 3′→5′ exonuclease resistant quencher domain and compositions for practicing the same

摘要

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.

主权项

1. 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 comprising a template nucleic acid, a nucleic acid polymerase having 3′→5′ exonuclease activity, and a FET labeled oligonucleotide comprising a target-binding sequence, a fluorophore, a nucleic acid intercalator, and a quencher, wherein said nucleic acid intercalator and said quencher are covalently bonded to the 3′ end of said FET labeled oligonucleotide; (b) subjecting said primer extension mixture to primer extension reaction conditions; and (c) detecting a fluorescent signal from said FET labeled oligonucleotide in said mixture, whereby a change in said fluorescent signal is indicative of the presence of a primer extension product in said mixture.