Real-time sequence determination

a single-molecule, sequence-based technology, applied in the direction of fluorescence/phosphorescence, enzymology, biomass after-treatment, etc., can solve the problem that the polymerase cannot add any additional bases to the end of the strand, the routine application of primer-walking for sequencing large dna fragments is limited, and the random approach is typically not sufficient to complete sequence determination. problems, to achieve the effect of enhancing interaction and enhancing interaction

US20050266424A1Inactive Publication Date: 2005-12-01HARDIN SUSAN +4
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Patent Information

Authority / Receiving Office
US · United States
Patent Type
Applications(United States)
Current Assignee / Owner
Publication Date
2005-12-01
Estimated Expiration
Not applicable · inactive patent

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Abstract

A sequencing methodology is disclosed that allows a single DNA or RNA molecule or portion thereof to be sequenced directly and in substantially real time. The methodology involves engineering a polymerase and / or dNTPs with atomic and / or molecular tags that have a detectable property that is monitored by a detection system.
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Description

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a single-molecule sequencing apparatus and methods. More particularly, the present invention relates to a single-molecule sequencing apparatus and methods using tagged polymerizing agents and / or tagged monomers where the tagged polymerizing agent and / or the tagged monomers undergo a change in a detectable property before, during and / or after monomer insertion into a growing polymer chain. The apparatus and methods are ideally-suited for sequencing DNA, RNA, polypeptide, carbohydrate or similar bio-molecular sequences under near real-time or real-time conditions. The present invention also relates to a single-molecule sequencing apparatus and methods using tagged depolymerizing agents and / or tagged depolymerizable polymer where the tagged depolymerizing agent and / or the tagged depolymerizable polymer undergo a change in a detectable property before, during and / or after monomer removal...

Examples

examples

Cloning and Mutagenesis of Tag Polymerase

[0245] Cloning Bacteriophage lambda host strain Charon 35 harboring the full-length of the Thermus aquaticus gene encoding DNA polymerase I (Taq pol I) was obtained from the American Type Culture Collection (ATCC; Manassas, Va.). Taq pol I was amplified directly from the lysate of the infected E. coli host using the following DNA oligonucleotide primers:

Taq Pol I forward5′-gc gaattc atgaggggga tgctgcccct ctttgagccc-3′Taq Pol I reverse5′-gc gaattc accctccttgg cggagcgc cagtcctccc-3′

[0246] The underlined segment of each synthetic DNA oligonucleotide represents engineered EcoRI restriction sites immediately preceding and following the Taq pol I gene. PCR amplification using the reverse primer described above and the following forward primer created an additional construct with an N-terminal deletion of the gene:

Taq Pol I_A293_trunk5′-aatccatgggccctggaggaggc cccctggcccccgc-3′

[0247] The underlined segment corresponds to an engineered NcoI res...