Nucleotides

• US 20070026447A1
• Filed: 08/30/2006
• Published: 02/01/2007
• Est. Priority Date: 05/19/1999
• Status: Abandoned Application

• Current Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Original Assignee: Cornell Research Foundation Incorporated (Cornell University)
• Inventors: Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Levene, Michael, Craighead, Harold, Webb, Watt
• Application Number:
  US11/513,760
• Publication Number:
  US 20070026447A1
• 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 charge-switch nucleotide phosphate (NP) probe, said NP probe comprising:

an intact NP probe having a terminal phosphate with a fluorophore moiety attached thereto via a linker carrying at least one positive charge, wherein said terminal phosphate is a pyrophosphate with a fluorophore moiety attached thereto, said intact NP probe having a first molecular charge associated therewith, whereupon cleavage of said terminal phosphate as a phosphate fluorophore moiety, said phosphate fluorophore moiety carries a second molecular charge, wherein the difference between said first molecular charge and said second molecular charge is at least 0.5.

63. A charge-switch nucleotide phosphate (NP) probe, said NP probe comprising:

an intact NP probe having a terminal phosphate with a fluorophore moiety attached thereto via a linker carrying at least two positive charges, wherein said terminal phosphate is a pyrophosphate with a fluorophore moiety attached thereto, said intact NP probe having a first molecular charge associated therewith, whereupon cleavage of said terminal phosphate as a phosphate fluorophore moiety, said phosphate fluorophore moiety carries a second molecular charge, wherein the difference between said first molecular charge and said second molecular charge is at least 0.5.

64. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, wherein said charge-switch NP probe is a nucleotide triphosphate (NTP) and said phosphate detectable moiety is a pyrophosphate with a fluorophore moiety attached thereto.

65. The intact charge-switch NP probe according to claim 64, wherein upon cleavage of said phosphate detectable moiety as a pyrophosphate fluorophore moiety, said pyrophosphate fluorophore moiety carries a positive charge relative to said intact NTP probe.

66. The intact charge-switch NP probe according to claim 64, wherein upon cleavage of said phosphate detectable moiety as a pyrophosphate fluorophore moiety, said pyrophosphate fluorophore moiety carries a negative charge relative to said intact NTP probe.

67. The intact charge-switch NP probe according to claim 64, wherein said fluorophore moiety is attached to said terminal phosphate via a linker.

68. The intact charge-switch NP probe according to claim 67, wherein said fluorophore linker is an alkylene group having a plurality of carbons.

69. The intact charge-switch NP probe according to claim 67, wherein said linker carries at least one positive charge.

70. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, and wherein said charge-switch NP probe is a member selected from the group consisting of a deoxynucleotide triphosphate (dNTP), and a nucleotide triphosphate (NTP).

71. The intact charge-switch NP probe according to claim 70, wherein said charge-switch NP probe is a deoxynucleotide triphosphate (dNTP).

72. The intact charge-switch NP probe according to claim 71, wherein said deoxynucleotide triphosphate (dNTP) is a member selected from the group consisting of deoxyadenosine triphosphate, deoxycytosine triphosphate, deoxyguanosine triphosphate, deoxythymidine triphosphate and deoxyuridine triphosphate.

73. The intact charge-switch NP probe according to claim 70, wherein said nucleotide triphosphate (NTP) is a member selected from the group consisting of adenosine triphosphate, cytosine triphosphate, guanosine triphosphate and uridine triphosphate.

74. An intact charge-switch nucleotide phosphate (NP) probe, wherein upon enzymatic cleavage-of said intact charge-switch NP probe to produce a phosphate detectable moiety, said phosphate detectable moiety migrates to an electrode, and said intact charge-switch NP probe migrates to the other electrode, wherein at least one of the phosphate moieties of said nucleotide phosphate probe has an ionized oxygen atom with a counter-cation associated therewith.

75. The intact charge-switch NP probe according to claim 74, wherein said counter-cation is a metal ion.

76. The intact charge-switch NP probe according to claim 75, wherein said metal ion is selected from the group consisting of Mg²⁺, K⁺, and Na⁺.

77. The intact charge-switch NP probe according to claim 76, wherein said counter-cation is a divalent cation.