SHORT CYCLE METHODS FOR SEQUENCING POLYNUCLEOTIDES

• US 20150292008A1
• Filed: 03/19/2015
• Published: 10/15/2015
• Est. Priority Date: 11/12/2003
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Fluidigm Corporation
• Inventors: Lapidus, Stanley N., HARRIS, TIMOTHY D., BUZBY, PHILIP R.
• Granted Patent: US 9,657,344 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 2527/113 Time

Abstract

The invention provides methods for sequencing a polynucleotide comprising stopping an extension cycle in a sequence by synthesis reaction before the reaction has run to near or full completion.

Claims

1. A method for obtaining sequence information from a plurality of different target nucleic acids immobilized at discrete locations on a surface, wherein a plurality of the target nucleic acids comprise a common adaptor sequence and have a primer hybridized thereto;

wherein the method comprises multiple cycles of the following steps;

(a) contacting the target nucleic acids with a solution that contains an enzyme and a detectably labeled nucleotide such that the enzyme catalyzes template-dependent addition of the labeled nucleotide to extend a plurality of the primers,

wherein the labeled nucleotide is not a chain-terminating nucleotide, and

wherein addition of the labeled nucleotide to a primer interferes with progression of the enzyme;

then

(b) detecting addition of the labeled nucleotide to at least some of the primers, thereby indicating the presence of a complementary nucleotide in the target nucleic acid hybridized thereto.

2. The method of claim 1, wherein the enzyme is a polymerase.

3. The method of claim 1, wherein the detectable label is a fluorescent label.

4. The method of claim 1, wherein the detectable label is a FRET donor or a FRET acceptor.

5. The method of claim 1, wherein the labeled nucleotide is selected in each cycle such that the target nucleic acids are sequentially contacted with dATP, dCTP, dGTP and either TTP or dUTP.

6. The method of claim 1, comprising removing the detectable label from nucleotides that have extended the primer following step (b) and before the next cycle.