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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

Inactive Publication Date: 2005-12-01
HARDIN SUSAN +4
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The present invention provides a system for detecting changes in interactions between a polymerizing or depolymerizing agent and its substrates in real-time or near real-time. The system uses a polymerizing or depolymerizing agent modified with a molecular or atomic tag located at or near a site on the agent, which undergoes a detectable property change before, during, and / or after monomer incorporation. The detectable property can be a change in the tag's fluorescence, intensity, frequency, or other property that can be measured using a detector. The system can be used with different types of polymerases and monomers, and can be used in various polymerization reactions."

Problems solved by technology

However, once the ddNTP is incorporated, the polymerase is unable to add any additional bases to the end of the strand.
However, this random approach is typically not sufficient to complete sequence determination, since gaps in the sequence often remain after computer assembly.
The necessity of designing and synthesizing new primers, coupled with the expense and the time required for their synthesis, has limited the routine application of primer-walking for sequencing large DNA fragments.
Conventional DNA sequencing strategies and methods are reliable, but time, labor, and cost intensive.
This requirement limits the length of sequence that can be determined, and increases the number of manipulations that must be performed before any sequence data is obtained.

Method used

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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...

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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.

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...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07H21/04C12M1/00C12N9/10C12N9/12C12N9/22G01N21/64C12N15/09C12P19/34C12Q1/68C12Q1/6869C12Q1/70G01N21/78G01N33/48G01N33/50G01N33/53G01N33/566G01N33/58
CPCC12Q1/6869C12Q2565/101C12Q2565/301C12Q2537/143C12Q2565/133
Inventor HARDIN, SUSANGAO, XIAOLIANBRIGGS, JAMESWILLSON, RICHARDTU, SHIAO-CHUN
Owner HARDIN SUSAN
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