Single molecule sequencing with two distinct chemistry steps

• US 9,587,275 B2
• Filed: 09/08/2015
• Issued: 03/07/2017
• Est. Priority Date: 02/18/2010
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Emig, Robin, Jia, Lei, Hanes, Jeremiah, Sebo, Lubomir
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/848,136
• Publication Number:
  US 20160010150A1
• Time in Patent Office: 546 Days
• Field of Search: 435/6.1, 435/91.1, 435/6.12
• US Class Current: 1/1
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/11 containing cyclic phosphate
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/213 containing cyclic phosphate
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2563/107 fluorescence

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.

Claims

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.

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 at least one of said plurality of nucleotide analogs is chosen from compounds having Formula IV:

7. The composition of claim 6 wherein L₁ and L₂ are members of a donor-quencher or a FRET pair.

8. The composition of claim 6 wherein B comprises A, T, G, C, U or I.

12. The method of claim 1, wherein the plurality of nucleotide analogs comprises at least four types of nucleotide analogs, each type comprising a unique base-pairing moiety and a unique fluorophore that is distinguishable from the fluorophores of the other types of nucleotide analogs of said plurality.

13. The method of claim 12, wherein the plurality of nucleotide analogs further comprises a nucleotide analog having a fluorescence quenching moiety that is removed from the nucleotide analog upon incorporation of the nucleotide analog into the nascent strand.

16. The method of claim 1 wherein the at least one residue that can be removed by an exonuclease activity comprises a nucleotide.

17. The method of claim 1 wherein the at least one residue that can be removed by an exonuclease activity comprises the structure:

18. The method of claim 17 wherein R₆ is a substituted or unsubstituted 5- or 6-membered unsaturated ring containing one, two, three, or four independently selected heteroatoms, independently selected from oxygen, nitrogen, and sulfur.

19. The method of claim 17 wherein R₆ is a substituted or unsubstituted, 2-, 3- or 4-pyridinyl, pyrazinyl, 2-, 4-, or 5-pyrimidinyl, pyridazinyl, triazolyl, tetrazolyl, imidazolyl, 2- or 3-thienyl, 2- or 3-furyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, quinolyl, isoquinolyl, benzimidazolyl, benzotriazolyl, benzofuranyl, or benzothienyl.

9. 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 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 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;

10. The composition of claim 9 wherein L₁ and L₂ are members of a donor-quencher or a FRET pair.

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

14. A method of determining a nucleotide base incorporated by a polymerase enzyme or enzyme complex in a nucleic add polymerization reaction, the method comprising the steps of:

(a) conducting a nucleic acid polymerization reaction that utilizes both 5′

to 3′

polymerization activity and 3′

to 5′

exonuclease activity of a polymerase enzyme or enzyme complex, and that results in production of a nascent strand in a template-dependent manner, wherein said reaction is conducted in the presence of;

(i) a template nucleic acid comprising a target nucleic acid sequence,

(ii) a primer nucleic acid comprising a sequence which is complementary to a region of the template nucleic acid,

(iii) a polymerase enzyme or an enzyme complex comprising 5′

to 3′

polymerization activity and 3′

to 5′

exonuclease activity,

(iv) a plurality of nucleotide analogs, wherein at least one nucleotide analog of said plurality comprises at least one base-pairing moiety, at least one residue that can be cleaved by an exonuclease activity, and at least one label moiety, said label moiety comprising a photo-detectable label,

wherein the label moiety of an individual nucleotide analog becomes incorporated into the nascent strand;

(b) detecting the photo-detectable label while the label moiety is coupled to the nascent strand, wherein said label is indicative of the identity of the base or bases present in the nucleotide analog incorporated by the enzyme or enzyme complex into the nascent strand; and

(c) after step (b), allowing the 3′

to 5′

exonuclease activity of the enzyme or enzyme complex to remove the labeled moiety from the nascent strand.

15. The method of claim 14, wherein said detecting step is performed during each successive nucleotide incorporation event after the photo-detectable label of the nucleotide analog incorporated in the previous incorporation event is removed via the 3′

to 5′

exonuclease activity of the enzyme or enzyme complex.