System and method for nucleic acid sequencing by polymerase synthesis

• US 8,530,154 B2
• Filed: 07/21/2006
• Issued: 09/10/2013
• Est. Priority Date: 12/14/1998
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Williams, John G. K.
• Primary Examiner(s): Whisenant, Ethan C
• Application Number:
  US11/459,182
• Publication Number:
  US 20090170074A1
• Time in Patent Office: 2,608 Days
• Field of Search: 435/6, 435/6.1, 536/23.1, 536/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C07D 209/12 Radicals substituted by oxygen atoms
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. 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 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

This invention relates to improved methods for sequencing and genotyping nucleic acid in a single molecule configuration. The method involves single molecule detection of fluorescent labeled PPi moieties released from NTPs as a polymerase extension product is created.

Claims

1. A method for sequencing a plurality of target nucleic acid molecules, the method comprising:

subjecting the plurality of target nucleic acid molecules to a polymerization reaction including a plurality of types of nucleotides or nucleotide analogs to yield a population of growing nucleic acid strands that are complementary to their respective target nucleic acid molecules, wherein the target nucleic acid molecule and/or a nucleic acid polymerase is attached to a support, where at least one of the types of nucleotides or nucleotide analogs includes a first atomic or molecular tag bonded to a nucleotide or nucleotide analog part released during incorporation; and

optically identifying a time sequence of incorporation events, at least one of the incorporation events involving the incorporation of a tagged nucleotide or nucleotide analog into

at least one growing nucleic acid strand in the population, where each event involving a tagged nucleotide or nucleotide analog is characterized by a change in a detectable property of the first tag wherein the tagged nucleotides or nucleotide analog types further include one additional tag or a plurality of additional tags.

2. The method of claim 1, wherein the additional tag is or the additional tags are attached to the nucleotides or nucleotide analogs at a base, sugar moiety, or phosphate.

3. The method of claim 1, wherein three or less of the plurality of types of nucleotides or nucleotide analogs have a different tag.

4. The method of claim 1, wherein the different types of nucleotides or nucleotide analogs have the same tag but are distinguished by different properties of the nucleotides or nucleotide analogs, and/or emission properties of the tags.