DNA polymerase

a polymerase and dna technology, applied in the field of dna polymerases, can solve the problems of limiting the use of unnatural or modified nucleotide bases and the applications they enable, limiting the substrate discrimination stringent, and preventing the efficient pcr amplification of damaged dna templates, etc., to achieve the effect of expanding the substrate range of such polymerases and high fidelity

Inactive Publication Date: 2007-01-25
MEDICAL RESEARCH COUNCIL
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AI Technical Summary

Benefits of technology

[0010] The present inventors modified the principles of directed evolution, (in particular compartmentalised self replication) described in GB97143002, 986063936 and GB 01275643 in the name of the present inventors, to relax the steric control of high fidelity DNA polymerases and consequently to expand the substrate range of such polymerases. All of the documents listed above are herein incorporated by reference.

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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  • DNA polymerase
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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0180] General Methods

List of primers:1:5′-CAG GAA ACA GCT ATG ACA AAA(SEQ ID NO: 3)ATC TAG ATA ACG AGG GA-3′;A.G mismatch2:5′-GTA AAA CGA CGG CCA GTA CCA(SEQ ID NO: 4)CCG AAC TGC GGG TGA CGCCAA GCC-3′; C.C mismatch3:5′-AAA AAT CTA GAT AAC GAG GGC(SEQ ID NO: 13)AA-3′4:5′-ACC ACC GAA CTG CGG GTG ACG(SEQ ID NO: 14)CCA AGC G-3′5:5′-GAA CTG CGG GTG ACG CCA AGC(SEQ ID NO: 15)GCA 3′; A.A mismatch6:5′-CC GAA CTG CGG GTG ACG CCA(SEQ ID NO: 16)AGC GG 3′; G.G mismatch7:5′-GAA CTG CGG GTG ACG CCA AGC(SEQ ID NO: 17)GCG-3′; G.A mismatch8:5′-AAA AAT CTA GAT AAC GAG GGC(SEQ ID NO: 18)AA-3′9:5′-CCG ACT GGC CAA GAT TAG AGA(SEQ ID NO: 19)GTA TGG-3′10:5′-GAT TTC CAC GGA TAA GAC TCC(SEQ ID NO: 20)GCA TCC-3′11:5′-GGG AGA CGA TGA TGC AGA TAA(SEQ ID NO: 21)CCA GAG C-3′12:5′-GCC GAT AGA TAG CCA CGG ACT(SEQ ID NO: 22)TCG TAG-3′13:5′-GGA GTA GAT GCT TGC TTT TCT(SEQ ID NO: 23)GAG CC-3′14:5′-GAG TTC GTG CTT ACC GCA GAA(SEQ ID NO: 24)TGC AG-3′15:5′-ACC GAA CTG CGG GTG ACG CCA(SEQ ID NO: 25)AGC G 3′16:5′-ACC G...

example 2

[0189] 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 (fOext) 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-...

example 3

[0190] 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 pyrmidine 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 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-fidelity DNA polymerases perform an astonishing feat of molecular recognition, incorporating ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C07H21/04C12P21/06C12P19/34C12N9/22C12N9/12C12N15/10
CPCC12N9/1252
Inventor HOLLIGER, PHILIPPGHADESSY, FARIDD'ABBADIE, MARC
Owner MEDICAL RESEARCH COUNCIL
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