Single molecule sequencing of captured nucleic acids

• US 7,666,593 B2
• Filed: 08/26/2005
• Issued: 02/23/2010
• Est. Priority Date: 08/26/2005
• Status: Active Grant

• Current Assignee: Fluidigm Corporation
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Lapidus, Stanley
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/213,621
• Publication Number:
  US 20070048744A1
• Time in Patent Office: 1,642 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• 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 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.

Claims

1. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:

capturing target nucleic acids with a sequence-specific capture probe to produce a target/probe duplex;

melting said target/probe duplex to release said target nucleic acids;

annealing a primer to the target nucleic acids to produce a target/primer duplex;

attaching said target/primer duplex to a surface such that at least a portion of said target/primer duplex is individually optically resolvable;

introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition;

removing unincorporated nucleotide; and

identifying nucleotide species incorporated into the extended primer, thereby obtaining sequence information from said target.

2. The method of claim 1, further comprising the step of removing said optically-detectable label from nucleotide incorporated into said primer.

3. The method of claim 1, wherein said capturing step comprises hybridizing said target nucleic acids to capture probes immobilized in a polyacrylamide gel.

4. The method of claim 1, wherein said capture probes are attached to a bead.

5. The method of claim 1, wherein said optically-detectable label is a fluorescent label.

6. The method of claim 1, wherein said introducing, removing, and identifying steps are repeated at least once.

7. The method of claim 1, wherein said surface comprises an epoxide.

8. The method of claim 1, wherein said attaching step comprises modifying said target nucleic acid to introduce a 3′

or 5′

amine.

9. The method of claim 1, wherein said surface is streptavidnated.

10. The method of claim 9, wherein said target and/or said primer comprise a biotin end label.

11. The method of claim 1, wherein the sequence specific-capture probes of multiple sequences are used.

12. The method of claim 1, wherein the target nucleic acids are produced as a result of fragmentation by sonication.