Patent ID: 9670243
Date: 2017-06-06
CPC Classifications: C07H,C12Q

Claim:
1. A method of determining the nucleotide sequence of a target nucleic acid sequence, comprising the steps of (a) providing a reaction complex comprising a template nucleic acid comprising a target nucleic acid sequence, a primer nucleic acid comprising a. sequence which is complementary to a region of the template nucleic acid, and a polymerase enzyme or an enzyme complex, which comprises 5′ to 3′ polymerization activity and 3′ to 5′ exonuclease activity; (b) contacting the reaction complex with a plurality of nucleotide analogs, Wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one non-complementary nucleotide residue and at least one label moiety comprising a photo-detectable label that is indicative of the identity of the base-pairing moiety, further wherein the at least one nucleotide analog of said plurality is chosen from compounds having formula I: or a pharmaceutically acceptable salt or hydrate thereof, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, or 9; R 1 and each R 2 are O − ; or R 1 is and each R 2 is O − ; or R 1 is O − , one R 2 , is and any remaining R 2 is independently O − , S − , BH 3 − , or CH 3 ; R 3 is chosen from: wherein: B X X each R Y F is a fluorescent dye; R Y L Q is a fluorescence quenching moiety; and B (c) allowing the enzyme or enzyme complex to incorporate a nucleotide analog in a template-dependent manner into a nascent strand via the enzyme's or enzyme complex's 5′ to 3′ polymerization activity, whereby the label moiety of the nucleotide analog is coupled to the nascent strand; (d) detecting the photo-detectable label of the incorporated nucleotide analog; (e) allowing the enzyme or enzyme complex to remove the label. moiety of the incorporated nucleotide analog from the nascent strand via the enzyme's or enzyme complex's 3′ to 5′ exonuclease activity; and (f) repeating steps (c)-(e) to determine the sequence of the target nucleic acid sequence.