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Thermostable or thermoactive dna polymerase with attenuated 3'-5' exonuclease activity

An exonuclease and thermostability technology, applied in the field of DNA polymerase, can solve the problems of time consumption and high price

Inactive Publication Date: 2004-03-17
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This combination technique can be used to achieve the desired amount of 3'-5' exonuclease activity, but it is expensive and must be optimized step by step, thus consuming time

Method used

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  • Thermostable or thermoactive dna polymerase with attenuated 3'-5' exonuclease activity
  • Thermostable or thermoactive dna polymerase with attenuated 3'-5' exonuclease activity
  • Thermostable or thermoactive dna polymerase with attenuated 3'-5' exonuclease activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0699] Example 1: Mutations of pCS6 and pCS5

[0700] This example describes site-directed mutagenesis of the 3'-5' exonuclease domain of a thermostable and heat-activated DNA polymerase.

[0701] In the first round of mutagenesis, site-directed mutagenesis was used to change the residues of thermostable DNA polymerase. These residues were either known to have an important role in coordinating divalent metal cations, the high-fidelity domain of the Klenow fragment The homologous residues in the active site are either located between these homologous residues. See Derbyshire et al., 1995, Methods In Enzymology 262:363-85, which is incorporated herein by reference in its entirety. The plasmid pCS6 encoding the mutant chimeric thermostable DNA polymerase CS6 is used, as shown in Figure 5. CS6 is a mutant of the chimeric thermostable DNA polymerase CS5, as shown in Figure 4. The N-terminal part of CS5 is 291 residues from the N-terminal of Thermus species Z05 (Thermus species Z05) DNA...

Embodiment 2

[0717] Example 2: Production and purification of mutant thermostable DNA polymerase

[0718] The plasmid obtained from the above ligation contains the λP LThe polymerase gene under the control of a promoter. The plasmid was used to transform a host cell (DG116, ATCC#53606) containing the cI857 (thermally unstable) lambda repressor. The DG116 cells transformed with the plasmid containing the wild-type or mutant polymerase gene were used to inoculate 10ml of standard shake flask medium (SFM) supplemented with ampicillin (100μg / ml); composed of glucose and vitamin B 1 , Casein hydrolysate and minimal medium), grow overnight at 30°C and 250rpm. Use 5ml overnight culture to inoculate 450ml SFM+Ampicillin medium and incubate at 30℃, 250rpm shaker until the culture OD 600 Reach 0.6-0.8. The culture was then transferred to 37°C and grown overnight at 250 rpm to obtain temperature-induced expression of polymerase. See, for example, U.S. Patent Nos. 5,079,352, 5,420,029, and 5,618,711. The ...

Embodiment 3

[0720] Example 3: Standard analysis of 3'-5' exonuclease activity

[0721] The following examples provide a standard analysis method for measuring the thermostability or heat-activated DNA polymerase 3'-5' exonuclease activity and the definition of 1 unit of 3'-5' exonuclease activity.

[0722] In order to compare the 3'-5' exonuclease activity of the CS5 mutant derivatives, the degradation of the carboxyfluorescein (FAM)-labeled single-stranded oligonucleotide substrate NJS40 was measured.

[0723] NJS40: FAM-GCGCTAGGGCTGGCAAGTGTAGCGGTCAC (SEQID NO: 80)

[0724] Standard analysis method (40μl system) in 50mM Tricine, pH8.3, 25mM KOAc, 5% w / v DMSO, 0.5mM Mn(OAc) 2 And 5% (volume ratio) enzyme storage buffer component (in the reaction solution: 1mM Tris, pH8.0, 0.005mM EDTA, 0.05mM DTT, 5mM KCl, 0.025% Tween 20, 2.5% glycerol) containing 4pmol (100 nM) EAM labeled single-stranded oligonucleotide NJS40. By adding NJS40 and Mn(OAc) 2 The reaction was initiated and incubated at 63°C f...

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Abstract

The present invention relates to thermostable or thermoactive DNA polymerases with attenuated 3'-5' exonuclease ("proofreading") activity, methods for their synthesis, methods for their use, and kits comprising them. The enzymes are useful in many recombinant DNA techniques, especially nucleic acid amplification by the polymerase chain reaction (PCR).

Description

Invention field [0001] The present invention relates to thermostable or heat-activated DNA polymerases with impaired 3'-5' exonuclease ("correction") activity, methods for their synthesis, methods for their application, and reagents containing them box. It is an enzyme used in many recombinant DNA technologies, especially nucleic acid amplification by polymerase chain reaction (PCR). Background of the invention [0002] DNA polymerase uses complementary template DNA strands and primers in the direction from 5'to 3'to sequentially increase nucleotides from deoxynucleoside triphosphates (nucleotides) at the 3'-hydroxyl free end of the extended strand to synthesize DNA molecules . The template strand determines the sequence of nucleotide increase by Watson-Crick base pairing. In cells, DNA polymerase is involved in DNA repair synthesis and replication. See, for example, Komberg et al., 1992, DNA synthesis, W.H. Freeman, New York; Alberts et al., 1994, Cellular and Molecular Biology,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N1/21C12N9/12C12N15/54
CPCC07K2319/00C12N9/1252
Inventor N·J·舍恩布伦纳T·W·迈尔斯D·H·格尔弗兰德
Owner F HOFFMANN LA ROCHE & CO AG
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