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DNA polymerase

a polymerase and dna technology, applied in the field of dna polymerases, can solve the problems of limiting preventing the efficient pcr amplification of damaged dna templates, and restricting the use of unnatural or modified nucleotide bases and the applications they enabl

Inactive Publication Date: 2009-12-10
UK RES & INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While desirable in nature, such stringent substrate discrimination is limiting for many applications in biotechnology.
Specifically, it restricts the use of unnatural or modified nucleotide bases and the applications they enable.
It also precludes the efficient PCR amplification of damaged DNA templates.
The disadvantage of the use of translesion synthesis polymerases for biotechnological uses is that they depend on cellular processivity factors for their activity, such as PCNA.
Moreover such polymerases are not stable at the temperatures at which certain biotechnological techniques are performed, such as PCR.
Furthermore most Translesion synthesis polymerases have a much reduced fidelity, which would severely compromise their utility for cloning.
However, such methods are complex, prone to error and are laborious.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

General Methods

List of Primers:

[0173]

 1:5′-CAG GAA ACA GCT ATG ACA AAA ATC TAG ATA ACG AGG GA-3′;(SEQ ID NO: 3)A•G mismatch 2:5′-GTA AAA CGA CGG CCA GTA CCA CCG AAC TGC GGG TGA CGC(SEQ ID NO: 4)CAA GCC-3′; C•C mismatch 3:5′-AAA AAT CTA GAT AAC GAG GGC AA-3′(SEQ ID NO: 13) 4:5′-ACC ACC GAA CTG CGG GTG ACG CCA AGC G-3′(SEQ ID NO: 14) 5:5′-GAA CTG CGG GTG ACG CCA AGC GCA 3′; A•A mismatch(SEQ ID NO: 15) 6:5′-CC GAA CTG CGG GTG ACG CCA AGC GG 3′; G•G mismatch(SEQ ID NO: 16) 7:5′-GAA CTG CGG GTG ACG CCA AGC GCG-3′; G•A mismatch(SEQ ID NO: 17) 8:5′-AAA AAT CTA GAT AAC GAG GGC AA-3′(SEQ ID NO: 18) 9:5′-CCG ACT GGC CAA GAT TAG AGA GTA TGG-3′(SEQ ID NO: 19)10:5′-GAT TTC CAC GGA TAA GAC TCC GCA TCC-3′(SEQ ID NO: 20)11:5′-GGC AGA CGA TGA TGC AGA TAA CCA GAG C-3′(SEQ ID NO: 21)12:5′-GCC GAT AGA TAG CCA CGG ACT TCG TAG-3′(SEQ ID NO: 22)13:5′-GGA GTA GAT GCT TGC TTT TCT GAG CC-3′(SEQ ID NO: 23)14:5′-GAG TTC GTG CTT ACC GCA GAA TGC AG-3′(SEQ ID NO: 24)15:5′-ACC GAA CTG CGG GTG ACG CCA AGC G 3′(SEQ ...

example 2

[0182]Kinetic analysis. Extension and incorporation kinetics of M1 and M4 for a selection of mismatches were measured using a gel-based steady-state kinetic assay (Goodman) (Tables 1 & 2). M1 and M4 respectively extend a C·C mispair 390 and 75-fold more efficiently than wtTaq. Examination of the other most disfavored mismatches (G·A, A·G, A·A, G·G) reveals generic, although less pronounced, increases of extension efficiencies, as suggested by the PCR assay (FIG. 4, FIG. 5). The gain in extension efficiency derives predominantly from increased relative Vmax values for the mutant polymerases, while Km for nucleotide substrates remains largely unchanged. For most DNA polymerases the relative efficiency of extending a given mispair (f0ext) is similar to the relative efficiency of forming it (finc) (Goodman 1993, Goodman 1990, Washington 2001). Indeed, M1 and M4 respectively incorporate dCTP opposite template base C 206- and 29-fold more efficiently than wtTaq (Table 2).

TABLE 2Steady-sta...

example 3

[0183]Translesion synthesis. Transversion mispairs represent distorting deviations from the cognate duplex structure. We therefore investigated if M1 and M4 were capable of processing other deviations of the DNA structure such as lesions in the template strand. Using a gel-extension assay we investigated their ability to traverse an abasic site and a cis-syn thymine pyrimidine dimer (CPD) template strand lesion. In control assays using an undamaged template, wtTaq, M1 and M4 efficiently and rapidly extended primers to the end of the template (FIG. 5). On the template containing an abasic site, wtTaq efficiently inserts a base opposite the lesion but, further extension is largely abolished. In contrast, both M1 and M4 are able to extend past the lesion and to the end of the template. The size of the final product is similar to that observed on the undamaged template indicating that bypass occurred without deletions. Perhaps the most striking example of the proficiency of M1 and M4 to...

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Abstract

The present invention relates to DNA polymerases. In particular the invention relates to a method for the generation of DNA polymerases exhibiting a relaxed substrate specificity. Uses of mutant polymerases produced using the methods of the invention are also described.

Description

RELATED APPLICATIONS[0001]This application is a divisional of U.S. application Ser. No. 11 / 417,403, which was filed on May 3, 2006, which is a continuation of Application No. PCT / GB04 / 004643, which was filed on 3 Nov. 2004, which designated the United States and was published in English, and which claims the benefit of United Kingdom Applications GB041087.8, filed 14 May 2004, and GB0325650.0, filed 3 Nov. 2003. The entire teachings of the above applications are incorporated herein by reference.FIELD OF INVENTION[0002]The present invention relates to DNA polymerases. In particular the invention relates to a method for the generation of DNA polymerases which exhibit a relaxed substrate specificity. Uses of engineered polymerases produced using the methods of the invention are also described.BACKGROUND[0003]Accurate DNA replication is of fundamental importance to all life ensuring the maintenance and transmission of the genome and limiting tumorigenesis in higher organisms. High-fidel...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12N9/00C12N15/11C12N15/00C12P19/34C12N15/87C12N9/12C12N15/10
CPCC12N9/1252
Inventor HOLLIGER, PHILIPPGHADESSY, FARIDD'ABBADIE, MARC
Owner UK RES & INNOVATION LTD
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