Single molecule sequencing with two distinct chemistry steps

Patent metadata

Field	Value
Publication number	US-2016010150-A1
Application number	US-201514848136-A
Country	US
Kind code	A1
Filing date	Sep 8, 2015
Priority date	Feb 18, 2010
Publication date	Jan 14, 2016
Grant date	—

Abstract

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.

First claim

What is claimed is:

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 an individual 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; (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 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.

2 . The method of claim 1 , wherein the photo-detectable label is a fluorophore.

3 . The method of claim 2 , wherein the nucleotide analog further comprises at least one fluorescence quenching moiety, and wherein the fluorescence quenching moiety is removed from the nucleotide analog upon incorporation of the nucleotide analog via the enzyme's or enzyme complex's 5′ to 3′ polymerization activity into the nascent strand.

4 . The method of claim 1 , wherein the at least one base-pairing moiety, having a 5′ end and a 3′ end, comprises one or more nucleotide residues that each comprises a base that is able to base pair with a corresponding base of the target nucleic acid sequence in an active site of the reaction complex.

5 . The method of claim 4 , wherein the 3′ end of the base-pairing moiety is connected to the label moiety via a phosphate linkage.

6 . The method of claim 1 , wherein the one or more non-complementary nucleotide residues are independently chosen from an abasic nucleotide residue and a nucleotide residue comprising a base which substantially lacks the ability to base pair with any of adenine, cytosine, guanine, thymine, or uracil.

7 . The method of claim 1 wherein at least one of said plurality of nucleotide analogs is chosen from compounds having Formula IV:

wherein B is a nucleobase; Q is H, OH, SH, or NHR; R 1 is selected from O and S; R 2 is selected from O, NH, NR, S, CH 2 , CRR′, CH2CH2, CRR′CRR′, C(O), CRNHR′, where R and R′ are independently selected from H, F, Cl, OH, NH2, methyl, ethyl, propyl, C 2 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, heterocycle, 4-pyridine, and 1-imidazone; U is a single bond,

R 3 is selected from —OR, —SR, —NRR′, or —CH 2 R; K 1 and K 2 are linker moieties; L 1 and L 2 are detectable labels; and n is from 0 to 6
wherein K 2 comprises a structure of Formula III.

wherein R 4 and R 5 , are independently selected from a bond to L 2 , H, OH, OR 7 , SR 7 or NHR 7 ; R 7 is H, methyl or a C 2 -C 6 alkyl group; and R 6 is either a bond to L 2 , a nucleobase, or an aryl, heteroaryl, or C 1 -C 6 aliphatic moiety.

8 . The composition of claim 7 wherein L 1 and L 2 are members of a donor-quencher or a FRET pair.

9 . The composition of claim 7 wherein B comprises A, T, G, C, U or I.

10 . 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; (c) allowing the enzyme or enzyme complex to incorporate a nucleotide analog in a template-dependent manner into a nascent strand via 5′ to 3′ polymerization activity of the enzyme or enzyme complex; (d) detecting a photo-detectable label moiety on 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 3′ to 5′ exonuclease activity of the enzyme or enzyme complex; and (f) repeating steps (c)-(e) to determine the sequence of the target nucleic acid sequence, wherein at least one of the plurality of nucleotide analogs is chosen from compounds having Formula IV:

wherein B is a nucleobase; Q is H, OH, SH, or NHR; R 1 is selected from O and S; R 2 is selected from O, NH, NR, S, CH 2 , CRR′, CH2CH2, CRR′CRR′, C(O), CRNHR′, where R and R′ are independently selected from H, F, Cl, OH, NH2, methyl, ethyl, propyl, C 2 -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, aryl, heterocycle, 4-pyridine, and 1-imidazone; U is a single bond,

R 3 is selected from —OR, —SR, —NRR′, or —CH 2 R; K 1 and K 2 are linker moieties; L 1 and L 2 are detectable labels; and n is from 0 to 6
wherein K 2 comprises a structure of Formula III.

wherein R 4 and R 5 , are independently selected from a bond to L 2 , H, OH, OR 7 , SR 7 or NHR 7 ; R 7 is H, methyl or a C 2 -C 6 alkyl group; and R 6 is either a bond to L 2 , a nucleobase, or an aryl, heteroaryl, or C 1 -C 6 aliphatic moiety.

11 . The composition of claim 10 wherein L 1 and L 2 are members of a donor-quencher or a FRET pair.

12 . The composition of claim 10 wherein B comprises A, T, G, C, U or I.

Assignees

• Pacific Biosciences California

Inventors

• Emig Robin
• Jia Lei
• Hanes Jeremiah
• Sebo Lubomir

Classifications

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2563/107

  fluorescence · CPC title

• C12Q2525/186

  incorporating a non-extendable or blocking moiety · CPC title

• C12Q2521/301

  Endonuclease · CPC title

• C12Q1/6869 Primary

  Methods for sequencing · CPC title