Method for sequencing nucleic acid molecules
First Claim
1. A method of sequencing a target nucleic acid molecule having a plurality of nucleotide bases comprising:
- providing a complex of a nucleic acid polymerizing enzyme and the target nucleic acid molecule oriented with respect to each other in a position suitable to add a nucleotide analog at an active site complementary to the target nucleic acid;
providing a plurality of types of nucleotide analogs proximate to the active site, wherein each type of nucleotide analog is complementary to a different nucleotide in the target nucleic acid sequence;
polymerizing a nucleotide analog at an active site, wherein the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid, leaving the added nucleotide analog ready for subsequent addition of nucleotide analogs;
identifying the nucleotide analog added at the active site as a result of said polymerizing; and
repeating said providing a plurality of types of nucleotide analogs, said polymerizing, and said identifying so that the sequence of the target nucleic acid is determined.
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Accused Products
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
61 Claims
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1. A method of sequencing a target nucleic acid molecule having a plurality of nucleotide bases comprising:
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providing a complex of a nucleic acid polymerizing enzyme and the target nucleic acid molecule oriented with respect to each other in a position suitable to add a nucleotide analog at an active site complementary to the target nucleic acid;
providing a plurality of types of nucleotide analogs proximate to the active site, wherein each type of nucleotide analog is complementary to a different nucleotide in the target nucleic acid sequence;
polymerizing a nucleotide analog at an active site, wherein the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid, leaving the added nucleotide analog ready for subsequent addition of nucleotide analogs;
identifying the nucleotide analog added at the active site as a result of said polymerizing; and
repeating said providing a plurality of types of nucleotide analogs, said polymerizing, and said identifying so that the sequence of the target nucleic acid is determined. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
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59. An apparatus suitable for sequencing a target nucleic acid molecule comprising:
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a support;
a nucleic acid polymerizing enzyme or oligonucleotide primer suitable to bind to a target nucleic acid molecule, wherein said nucleic acid polymerizing enzyme or said oligonucleotide primer is positioned on said support; and
a microstructure defining a confined region containing said support and said nucleic acid polymerizing enzyme or said oligonucleotide primer and configured to permit labeled nucleotide analogs that are not positioned on the support to move rapidly through the confined region. - View Dependent Claims (60)
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61. An apparatus suitable for sequencing a target nucleic acid molecule comprising:
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a support;
a nucleic acid polymerizing enzyme or oligonucleotide primer suitable to hybridize to a target nucleic acid molecule, wherein said nucleic acid polymerizing enzyme or said oligonucleotide primer is positioned on said support;
a housing defining a confined region containing said support and said nucleic acid polymerizing enzyme or said oligonucleotide primer and constructed to facilitate identification of labeled nucleotide analogs positioned on the support; and
optical waveguides proximate to the confined region to focus activating radiation on the confined region and to collect radiation from the confined region.
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Specification