Methods for analyzing nucleic acid sequences
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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.
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Citations
79 Claims
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1-61. -61. (canceled)
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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. - View Dependent Claims (63, 64, 65, 66, 67, 68, 69)
- locating the respective positions of a plurality of target nucleic acid template molecules in a field of immobilized polynucleotides;
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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. - View Dependent Claims (71, 72, 73, 76, 77, 78, 79)
- (a) obtaining an array of support-bound target nucleic acids in which each of said support-bound nucleic acids is individually optically resolvable;
- 74. The method of 73, wherein said inhibition is reversible.
Specification