Single molecule sequencing with two distinct chemistry steps

• US 9,447,464 B2
• Filed: 11/05/2013
• Issued: 09/20/2016
• 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:
  US14/072,343
• Publication Number:
  US 20140127680A1
• Time in Patent Office: 1,050 Days
• Field of Search: 435/6.1, 435/612, 435/91.1
• 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 for sequencing a nucleic acid template comprising:

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

wherein each type of nucleotide analog has a labeled leaving group that is cleavable by an exonuclease, and each type of nucleotide analog has a different label, wherein the labeled leaving group blocks further nucleotide incorporations at the 3′

hydroxyl position of the nucleotide analog once it is incorporated; and

(iv) an exonuclease activity which cleaves a labeled leaving group from an incorporated nucleotide analog;

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

a) a nucleotide associates with the polymerase, b) the nucleotide analog is incorporated, then c) the labeled leaving group on that nucleotide analog is cleaved by the exonuclease activity, thereby making the 3′

hydroxyl group of the incorporated nucleotide analog available for a subsequent nucleotide analog addition; and

detecting light from the labels while nucleic acid synthesis is occurring, and using light detected in the time between step a) in which the nucleotide analog associates with the polymerase, and step c) in which the labeled leaving group is cleaved to determine a sequence of the template nucleic acid.

2. The method of claim 1 wherein the polymerase enzyme comprises the exonuclease activity.

3. The method of claim 1 wherein the exonuclease 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.

4. The method of claim 1 wherein the exonuclease activity cleaves an incorporated nucleotide analog at a rate that is at least 100 times greater than rate of cleavage of an unincorporated nucleotide analog.

5. The method of claim 1 wherein the exonuclease activity cleaves an incorporated nucleotide analog at a rate that is at least 1000 times greater than rate of cleavage of an unincorporated nucleotide analog.

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

7. The method of claim 6 wherein multiple single polymerase enzymes are immobilized on the substrate, and sequencing from each polymerase enzyme is monitored concurrently.

8. The method of claim 1 wherein the template nucleic acid is immobilized on a substrate.

9. The method of claim 8 wherein multiple single nucleic acid templates are immobilized on the substrate, and sequencing from each polymerase enzyme is monitored concurrently.

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

11. 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.

12. The method of claim 1 wherein the template nucleic acid comprises DNA or RNA or a DNA/RNA hybrid.

13. The method of claim 1 wherein the label comprises a fluorescent label.

14. The method of claim 13 wherein the fluorescent label comprises a fluorescent dye or a fluorescent particle.