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DNA polymerases having modified nucleotide binding site for DNA sequencing

A technology of polymerase and DNA molecules, applied in the direction of DNA / RNA fragments, recombinant DNA technology, enzymes, etc., can solve the problems of reducing the accuracy of DNA sequences, reducing sensitivity, and difficulties

Inactive Publication Date: 2007-09-05
PRESIDENT & FELLOWS OF HARVARD COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, this reduces the sensitivity of the method, which is limited by the ability to detect bands with minimal signal
Second, this creates difficulties in determining whether a band of weak signal is a true signal from the addition of chain terminators, or an artifact caused by a pause site on the DNA where the polymerase dissociates
Third, the accuracy of DNA sequence determination in immediately adjacent bands is reduced because strong signals from one band mask weaker signals from adjacent bands
More importantly, although the overall degree of discrimination of these enzymes such as Klenow and Taq DNA polymerase was reduced, specific fragment intensities differed by more than 4-fold due to high discrimination at specific sequences in DNA

Method used

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  • DNA polymerases having modified nucleotide binding site for DNA sequencing
  • DNA polymerases having modified nucleotide binding site for DNA sequencing
  • DNA polymerases having modified nucleotide binding site for DNA sequencing

Examples

Experimental program
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Effect test

Embodiment 1D

[0222] Mutagenesis and Mutants of Example 1 DNA Polymerase Gene

[0223] Overproduction of DNA polymerase

[0224] The mutant DNA polymerase gene is cloned and expressed using standard techniques. Starting material for generating mutants of E. coli DNA polymerase I and Taq DNA polymerase - encoding a large fragment of E. coli DNA polymerase I (Klenow fragment) and a large fragment of Taq DNA polymerase (KlenTaq or Taq DNA polymerase, See Barnes 112 Gene 29, 1992 or Stoffel Fragment, see Lawyer et al. 2 PCR Methods Applied 275, 1993) the gene is expressed under the control of the T7 RNA polymerase promoter. Starting material for generation of mutants of T7 DNA polymerase - the gene encoding the 28 amino acid deletion of T7 DNA polymerase (see Tabor and Richardson, 264 J. Biol. Chem. 6447, 1989) is essential for the production of T7 DNA polymerase for sustained DNA synthesis. The factor E. coli thioredoxin (Tabor and Richardson, supra) was expressed under the control of...

Embodiment 2

[0227] Example 2 Quickly screen out the efficiency of incorporation of dideoxynucleotides from DNA polymerase

[0228] Mutants with increased efficiency of incorporation of deoxynucleotides

[0229] The mutant DNA polymerases were screened for their ability to incorporate dideoxynucleotides by SDS activity gel assay. This procedure is a modification of the method described in Spanos and Hübscher 91 Enzymology and Karawya et al 135 Analytical Biochem 318,1983. Briefly, 10 ml of cells were induced for 4 to 6 hours and then pelleted. Cell pellets were resuspended in 0.3ml 25mM Tris Hcl, pH7.0, 5mM EDTA. Mix 20μl of resuspended cells with 40μl of 1% SDS (sodium dodecylsulfonate), 2% mercaptoethanol, 30% glycerol, 0.04 % bromophenol blue and 100 mM Tris HCl, pH 6.8 solution were mixed. The mixture was incubated at 37°C for 5 minutes, and two 20 μl aliquots were applied to two SDS polyacrylamide gels. The separation gel of SDS polyacrylamide contains 8% polyacrylamide, 0.27...

Embodiment 3

[0236] Example 3 Single-stranded M13 DNA-5' 32 P-labeled 40mer

[0237] Preparation and purification of primer complexes

[0238] The template was 9950 nucleotides long M13 mGP1-2 single-stranded DNA as described in US Patent 4,795,699 (Figure 9). The deposit number of phage M13 mGP1-2 in ATCC is 40303. M13 mGP1-2 single-stranded DNA was purified as described by Tabor et al. 262 J Biol Chem., 16212, 1987 . Briefly, phage were purified by twice CsCl gradient centrifugation, dialyzed to remove CsCl, and DNA (human phage) was extracted with phenol:chloroform and 0.1% sodium dodecylsulfate. HCl, pH 7.5, dialyzed fully with 2mM EDTA, store at 4°C. Determine the concentration of M13 mGP1-2 single-stranded DNA with a photometer, the extinction coefficient is 8.1, and the A260 unit is equal to 1 mg / ml or 0.3 pmol per microliter M13 mGP1-2 template molecular.

[0239] The primer was a 40-mer synthesized by standard methods with the sequence 5'd(TTTTCCCAGTCACGACGTTGTAAAACGAC...

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Abstract

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. <IMAGE>

Description

[0001] This application is a continuation-in-part of Tabor and Richardson, entitled "DNA Polymerases with Modified Nucleotide Binding Sites for DNA Sequencing," filed October 17, 1994, the latter in its entirety (including the accompanying drawings ) is hereby incorporated by reference. Background of the invention [0002] This invention was made with government support, including US Department of Energy Contract No. DE-FG02-88ER60688. The US Government has certain rights in this invention. [0003] The present invention relates to DNA polymerases suitable for DNA sequencing and to automated and manual methods of DNA sequencing. [0004] The following is a brief overview of the field related to DNA sequencing technology. It is provided for general guidance to the applicant reading this application only and does not imply that any art referred to or referred to herein, directly or indirectly, is prior art to the appended claims. [0005] DNA sequencing generally involves gen...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/11C12Q1/68
CPCC12Q1/686C12N9/1252C12Q1/6869
Inventor S·泰伯C·C·里查尔德森
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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