Methods for analyzing nucleic acid sequences

• US 20060154288A1
• Filed: 01/19/2006
• Published: 07/13/2006
• Est. Priority Date: 05/19/1999
• Status: Active Grant

• 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.
• Granted Patent: US 7,943,307 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 deriving nucleotide sequence information from individual polynucleotide molecules, the method comprising the steps of:

locating the respective positions of a plurality of target nucleic acid template molecules in a field of immobilized polynucleotides;

exposing said field to a polymerase and at least one nucleotide comprising a detectable label under conditions that allow said nucleotide to be incorporated in a primer hybridized to said template molecule if said nucleotide is complementary to a nucleotide in said template immediately 5′

of said primer;

determining the position or positions of optical signal emanating from label attached to a nucleotide that has been incorporated in said primer; and

recording the identity and position of an incorporated nucleotide based upon said optical signal.

63. The method of claim 62, further comprising the step of compiling one or more nucleic acid sequences at one or more of said locations.

64. The method of claim 62, wherein said exposing step comprises incorporating no ore than one nucleotide on average per primer prior to said determining step.

65. The method of claim 62 wherein said locating step comprises randomly depositing said target nucleic acid template molecules onto said field.

66. The method of claim 62, wherein said target nucleic acid template molecules are fixed on said field by a chemical linkage.

67. The method of claim 66, wherein said chemical linkage is a biotin/avidin or a biotin/streptavidin linkage.

68. The method of claim 62, wherein said field is arrayed on a surface comprising glass or fused silica.

69. The method of claim 62, further comprising the step of neutralizing said label after said determining step.

70. A method for identifying one or more nucleotides in a nucleic acid target molecule, the method comprising the steps of:

(a) obtaining an array of support-bound target nucleic acids in which each of said support-bound nucleic acids is individually optically resolvable;

(b) exposing said array to a polymerase, a primer capable of hybridizing to at least some of said support-bound nucleic acids and at least one nucleotide comprising a detectable label under conditions that allow said nucleotide to be incorporated into said primer if said nucleotide is complementary to a nucleotide in said target immediately 5′

to said primer;

(c) processing said array in order to remove unincorporated nucleotides;

(d) determining the presence and position of label attached to an incorporated nucleotide;

(e) neutralizing any label observed in said determining step;

(f) repeating steps a, b, c, and d;

(g) identifying label present at any position in which presence of label has previously been determined in said determining step;

(h) repeating steps a, b, c, d, and e; and

(i) identifying a sequencing of nucleotides in said target nucleic acid based upon the sequence of labels detected at any given position in said array.

71. The method of claim 70, wherein said label is optically detectable.

72. The method of claim 71, wherein said label is a fluorescent label.

73. The method of claim 70, wherein said label present in an incorporated nucleotide, inhibits incorporation of a subsequent nucleotide at a position immediately 5′

of said incorporated nucleotide.

76. The method of claim 70, wherein said label is an acceptor fluorophore, and wherein a primer/polymerase complex comprises a donor fluorophore label attached thereto.

77. The method of claim 70, wherein said label is an acceptor fluorophore and wherein said polymerase comprises a donor fluorophore attached thereto.

78. The method of claim 71, wherein said identifying step comprises visualizing said label using an optical microscope.

79. The method of claim 78, wherein said optical microscope comprises a total internal reflection objective.

74. The method of 73, wherein said inhibition is reversible.

75. The method of claim 74, further comprising the step of cleaving label present on an incorporated nucleotide after said first identifying step.