Title: Method for sequencing nucleic acid molecules

Abstract

The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog beingmore » added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined. « less

Inventors:

Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

Issue Date:

2006-06-06

Research Org.:

Cornell Univ., Ithaca, NY (United States)

Sponsoring Org.:

USDOE

OSTI Identifier:

1175774

Patent Number(s):

7056661

Application Number:

09/572,530

Assignee:

Cornell Research Foundation, Inc. (Ithaca, NY)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10S - TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

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C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6869 - Methods for sequencing
C12Q1/6874 - involving nucleic acid arrays, e.g. sequencing by hybridisation
C12Q2521/543 - Immobilised enzyme
C12Q2537/149 - Sequential reactions
C12Q2561/113 - Real time assay
C12Q2561/12 - Fluorescence lifetime measurement
C12Q2565/518 - characterised by the immobilisation of the nucleic acid sample or target
C12Q2565/537 - characterised by the capture oligonucleotide acting as a primer

Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10S - TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

Y10S436/80 - Fluorescent dyes, e.g. rhodamine
Y10S436/805 - Optical property

Y - NEW / CROSS SECTIONAL TECHNOLOGIES Y10 - TECHNICAL SUBJECTS COVERED BY FORMER USPC Y10T - TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION

Y10T436/143333 - Saccharide [e.g., DNA, etc.]

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Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES