Nucleic acid analysis using terminal-phosphate-labeled nucleotides

• US 20050186619A1
• Filed: 03/25/2005
• Published: 08/25/2005
• Est. Priority Date: 05/19/1999
• Status: Active Grant

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Granted Patent: US 7,361,466 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2537/149 Sequential reactions
• CPC Class Codes: C12Q 2561/113 Real time assay
• CPC Class Codes: C12Q 2561/12 Fluorescence lifetime measurement
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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 being 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.

Claims

1-61. -61. (canceled)

62. A method of detecting the presence of a nucleic acid sequence comprising the steps of:

(a) conducting a nucleic acid polymerase reaction in the presence of at least one terminal-phosphate-labeled nucleotide having at least three phosphate groups in the polyphosphate chain, which reaction results in the production of a labeled polyphosphate; and

(b) detecting said labeled polyphosphate.

63. The method of claim 62 wherein step (a) further comprises conducting said polymerase reaction in the presence of at least two or more terminal-phosphate-labeled nucleotides with distinct labels.

64. The method of claim 62 wherein the at least one terminal-phosphate-labeled nucleotide is represented by the formula:

Base unit-sugar moiety-Y—

(P)_n—

P-Label moiety

wherein the base unit is selected from the group consisting of adenosine, thymidine, cytidine and guanosine base unit,

wherein p is a phosphate group and n is an integer of at least 2,

wherein y is an oxygen or sulfur atom;

65. The method of claim 64, wherein n is 2.

66. The method of claim 64 wherein the label moiety is linked to the phosphate via an NH, O, S, or a linker.

67. The method of claim 64 wherein the label moiety is a fluorescent moiety.

68. The method of claim 64 wherein the label moiety is selected from the group consisting of chromophores, enzymes, antigens, heavy metals, magnetic probes, dyes, phosphorescent groups, radioactive materials, chemiluminescent moieties, scattering or fluorescent nanoparticles, Raman signal generating moieties, and electrochemical detection moieties.