Single molecule sequencing with two distinct chemistry steps

• US 8,603,741 B2
• Filed: 02/08/2011
• Issued: 12/10/2013
• 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): WOOLWINE, SAMUEL C
• Application Number:
  US13/022,926
• Publication Number:
  US 20110212437A1
• Time in Patent Office: 1,036 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 for sequencing a nucleic acid template comprising:

providing a sequencing mixture comprising a polymerase enzyme, a template nucleic acid, a primer, a polymerase regent solution having the components for carrying out template directed synthesis of a growing nucleic acid strand, a plurality of types of nucleotide analogs;

wherein each type of nucleotide analog has a labeled leaving group, each with a different label, that blocks further nucleotide incorporations at the 3′

hydroxyl position of the incorporated nucleotide analog; and

a selective cleaving activity which more effectively cleaves a leaving group from an incorporated nucleotide analog than from an unincorporated nucleotide analog;

carrying out nucleic acid synthesis such that a plurality of nucleotide analogs are added sequentially whereby;

i) a nucleotide analog is incorporated, then ii) the labeled leaving group on that nucleotide analog is cleaved by the selective cleaving activity, making the 3′

hydroxyl group available for a subsequent nucleotide analog addition; and

detecting light from the labels while the polymerase reaction is occurring to determine a sequence of the template nucleic acid.

2. The method of claim 1 wherein the selective cleaving activity comprises an enzyme activity.

3. The method of claim 2 wherein the enzyme activity comprises exonuclease, esterase, or phosphatase activity.

4. The method of claim 2 wherein the enzyme activity is an activity that cleaves a phosphate, or an ester linkage.

5. The method of claim 1 wherein the selective cleaving activity cleaves an incorporated nucleotide analog at a rate that is at least 10 times greater than rate of cleavage of an unincorporated nucleotide analog.

6. The method of claim 1 wherein the selective cleaving activity selectively cleaves the labeled leaving group while the nucleotide analog is associated with the polymerase enzyme.

7. The method of claim 6 wherein the labeled leaving group is cleaved while the nucleotide analog is associated with the polymerase enzyme at a rate that is at least 10 times greater than rate of cleavage before the nucleotide analog is incorporated.

8. The method of claim 1 wherein the polymerase enzyme is immobilized on a substrate.

9. The method of claim 1 wherein the sequencing reaction occurs within an optical confinement on a surface.

10. The method of claim 1 wherein the plurality of types nucleotide analogs is four types of nucleotide analogs having bases corresponding to A, G, C, and T, or A, C, G, and U.

11. The method of claim 1 wherein the labeled leaving groups are attached to the oxygen at the 3′

hydroxyl of the nucleotide analogs.