Method for sequencing nucleic acid molecules
First Claim
1. A method of obtaining nucleotide sequence information for a target nucleic acid molecule having a plurality of nucleotide bases, said method comprising:
- providing a single 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, wherein the complex is immobilized to a solid support;
providing a plurality of types of nucleotide analogs that lack a blocking substituent proximate to the active site, each type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid and having a different detectable moiety, and polymerizing a nucleotide analog at the active site, wherein the added nucleotide analog is complementary to the nucleotide of the target nucleic acid and is capable of polymerization with a subsequent nucleotide analog;
identifying the nucleotide analog added at the active site as a result of said polymerizing by detecting its detectable label, wherein said identifying is carried out by directing activating radiation to a region corresponding to the active site and detecting the detectable label from the nucleotide analog added to the active site by a technique which reduces background noise resulting from free unpolymerized nucleotide analogs; and
repeating, with the complex immobilized to the solid support, said providing a plurality of types of nucleotide analogs, said polymerizing, and said identifying, so that nucleotide analogs added at downstream active sites are identified and, as a result, nucleotide sequence information for the target nucleic acid is determined.
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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.
564 Citations
40 Claims
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1. A method of obtaining nucleotide sequence information for a target nucleic acid molecule having a plurality of nucleotide bases, said method comprising:
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providing a single 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, wherein the complex is immobilized to a solid support; providing a plurality of types of nucleotide analogs that lack a blocking substituent proximate to the active site, each type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid and having a different detectable moiety, and polymerizing a nucleotide analog at the active site, wherein the added nucleotide analog is complementary to the nucleotide of the target nucleic acid and is capable of polymerization with a subsequent nucleotide analog; identifying the nucleotide analog added at the active site as a result of said polymerizing by detecting its detectable label, wherein said identifying is carried out by directing activating radiation to a region corresponding to the active site and detecting the detectable label from the nucleotide analog added to the active site by a technique which reduces background noise resulting from free unpolymerized nucleotide analogs; and repeating, with the complex immobilized to the solid support, said providing a plurality of types of nucleotide analogs, said polymerizing, and said identifying, so that nucleotide analogs added at downstream active sites are identified and, as a result, nucleotide sequence information for 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)
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Specification