Uses of terminal-phosphate-labeled nucleotides

• US 20050208557A1
• Filed: 03/25/2005
• Published: 09/22/2005
• Est. Priority Date: 05/19/1999
• Status: Abandoned Application

• Current Assignee: Harold G. Craighead, Jonas Korlach, Mathieu Foquet, Michael Levene, Stephen Turner, Watt W. Webb
• Original Assignee: Harold G. Craighead, Jonas Korlach, Mathieu Foquet, Michael Levene, Stephen Turner, Watt W. Webb
• Inventors: Craighead, Harold G., Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Webb, Watt W.
• Application Number:
  US11/089,822
• Publication Number:
  US 20050208557A1
• 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 a target using a labeled substrate or substrate analog comprising reacting said substrate or substrate analog in an enzyme-catalyzed reaction which produces a labeled polyphosphate that has independently detectable signal when said substrate or substrate analog reacts, and wherein, when the target is a nucleic acid, the labeled polyphosphate has at least two phosphates.

63. The method of claim 62 wherein the target is a nucleic acid target.

64. The method of claim 62 wherein the labeled substrate analog is a nucleotide analog comprising a terminal-phosphate labeled nucleoside polyphosphate.

65. The method of claim 62 wherein said enzyme-catalyzed reaction is nucleic acid polymerization.

66. The method of claim 65 wherein the nucleic acid polymerization is catalyzed by a nucleic acid polymerizing enzyme.

67. The method of claim 66 wherein the polymerizing enzyme is selected from the group consisting of a DNA polymerase, an RNA polymerase, and a reverse transcriptase.

68. The method of claim 62 wherein the said enzyme-catalyzed reaction is catalyzed by thermostable polymerase.

69. The method of claim 62, wherein the labeled polyphosphate contains 2 phosphate groups.

70. The method of claim 62 wherein the said labeled polyphosphate is represented by the formula:

Fluorescent moiety —

(P)_n—

P

wherein P is a phosphate and n is an integer of at least one.

71. The method of claim 70 wherein the fluorescent moiety is linked to the phosphate via an NH, O, S, or a linker.

72. The method of claim 70 wherein the labeled polyphosphate is represented by the formula

73. A method of detecting a target nucleic acid sequence using a labeled nucleotide analog comprising incorporating said analog by a nucleic acid polymerase into a polynucleotide complimentary to the target sequence thereby, producing a molecule which is neither pyrophosphate nor a mono-substituted pyrophosphate that undergoes a chemical change to produce a detectable signal.

74. The method of claim 73 wherein said chemical change is catalyzed by one or more enzymes.

75. The method of claim 73 wherein the said molecule is a labeled polyphosphate with at least two phosphate groups.

76. The method of claim 75 wherein the said labeled polyphosphate is represented by the formula:

Fluorescent moiety —

(P)_n—

P

wherein P is a phosphate and n is an integer of at least 1.

77. The method of claim 76 wherein the fluorescent moiety is linked to the phosphate via an NH, O, S, or a linker.

78. The method of claim 75 wherein the labeled polyphosphate is represented by the formula