Single molecule sequencing with two distinct chemistry steps

摘要

Methods, Compositions, and Systems are provided for nucleic acid sequencing where the sequential incorporation of nucleotides uses two distinct chemical steps. A plurality of nucleotide analogs, each having a labeled leaving group at its 3′ hydroxyl can be sequentially added to a growing strand in the presence of a selective cleaving activity that cleaves the 3′ hydroxyl leaving group preferentially after it has been incorporated. The selective cleaving agent can comprise an exonuclease activity, and the exonuclease activity can be a polymerase-associated exonuclease activity. Nucleotide analogs having labels on both a cleavable polyphosphate portion and on a 3′ hydroxyl leaving group can provide signals characteristic of nucleotide analog incorporation. Systems having illumination optics, collection optics, and substrates observe signals from the labels as they are being incorporated into a growing nucleic acid strand, allowing for the sequencing of template nucleic acids.

主权项

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 individual nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one residue that can be removed by an exonuclease activity, and at least one label moiety comprising a photo-detectable label that is indicative of the identity of the base-pairing moiety; (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 while the label moiety is coupled to the nascent strand; (e) after step (d), 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.