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Title: Methods for sequencing GC-rich and CCT repeat DNA templates

Abstract

The present invention is directed to a PCR-based method of cycle sequencing DNA and other polynucleotide sequences having high CG content and regions of high GC content, and includes for example DNA strands with a high Cytosine and/or Guanosine content and repeated motifs such as CCT repeats.

Inventors:
Publication Date:
Research Org.:
Los Alamos National Security, LLC, Los Alamos, NM (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1176115
Patent Number(s):
7,179,602
Application Number:
10/656,358
Assignee:
Los Alamos National Security, LLC (Los Alamos, NM) LANL
DOE Contract Number:
FG02-98ER62647
Resource Type:
Patent
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Robinson, Donna L. Methods for sequencing GC-rich and CCT repeat DNA templates. United States: N. p., 2007. Web.
Robinson, Donna L. Methods for sequencing GC-rich and CCT repeat DNA templates. United States.
Robinson, Donna L. Tue . "Methods for sequencing GC-rich and CCT repeat DNA templates". United States. doi:. https://www.osti.gov/servlets/purl/1176115.
@article{osti_1176115,
title = {Methods for sequencing GC-rich and CCT repeat DNA templates},
author = {Robinson, Donna L.},
abstractNote = {The present invention is directed to a PCR-based method of cycle sequencing DNA and other polynucleotide sequences having high CG content and regions of high GC content, and includes for example DNA strands with a high Cytosine and/or Guanosine content and repeated motifs such as CCT repeats.},
doi = {},
journal = {},
number = ,
volume = ,
place = {United States},
year = {Tue Feb 20 00:00:00 EST 2007},
month = {Tue Feb 20 00:00:00 EST 2007}
}

Patent:

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  • A method for sequencing DNA by hybridization that includes the following steps: forming an array of oligonucleotides at such concentrations that either ensure the same dissociation temperature for all fully complementary duplexes or allows hybridization and washing of such duplexes to be conducted at the same temperature; hybridizing said oligonucleotide array with labeled test DNA; washing in duplex dissociation conditions; identifying single-base substitutions in the test DNA by analyzing the distribution of the dissociation temperatures and reconstructing the DNA nucleotide sequence based on the above analysis. A device for carrying out the method comprises a solid substrate and a matrixmore » rigidly bound to the substrate. The matrix contains the oligonucleotide array and consists of a multiplicity of gel portions. Each gel portion contains one oligonucleotide of desired length. The gel portions are separated from one another by interstices and have a thickness not exceeding 30 .mu.m.« less
  • A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.
  • Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.
  • This patent describes a process for producing cloned, circular DNA molecules containing shortened target DNA fragments, the fragments derived from a long target DNA segment. The cloned, circular DNA molecules suitable for use in determining the nucleotide sequence of the long target DNA segment. The process consists the steps of: producing, by molecular cloning, double-stranded circular recombinant DNA molecules, each molecule containing vector DNA, a sequencing primer binding site, and a DNA region comprising a long target DNA segment, a first restriction site adjacent to the long target DNA segment adapted to be cut by a first restriction endonuclease inmore » a manner that creates a first terminus on the DNA molecules adjacent the long target DNA segment that is susceptible to digestion by an exonuclease, and a second restriction site located between the first restriction site and the sequencing primer binding site adapted to be cut by a second restriction endonuclease in a manner that creates, without additional terminus blocking or digestion, a second terminus on the DNA molecules that is not susceptible to digestion by an exonuclease; cutting the double-stranded circular recombinant DNA molecules at the first restriction site using a first restriction endonuclease and at the second restriction site using a second restriction endonuclease to form double-stranded linear recombinant DNA molecules having a first terminus that is susceptible to digestion by an exonuclease and a second terminus that is not susceptible to digestion by an exonuclease.« less
  • This patent describes a reagent kit for producing shortened DNA fragments useful for sequencing a target DNA segment. It comprises: a purified exonuclease reagent capable of unidirectionally digesting nucleotide bases asynchronously from a target DNA segment; and a purified recombinant vector molecule comprising vector DNA, a sequencing primer binding site, and a poly-linker region comprising first, second, and third restriction endonuclease sites. The first restriction site being capable of receiving by ligation the target DNA segment. The second and third restriction sites being located between the first restriction site and the sequencing primer binding site, and the second and thirdmore » restriction sites being so arranged that cleavage of the polylinker region at the second and third restriction sites will produce a linearized vector molecule having a first terminus adjacent the target DNA segment that is susceptible to digestion by the exonuclease reagent and a second terminus, not susceptible to digestion by the exonuclease reagent adjacent the sequencing primer binding site.« less