Alcohol-Mediated Error-Prone PCR

Claveau, S.; Sasseville, Maxime; Beauregard, Marc

doi:10.1089/dna.2004.23.789

Cited by 13 publications

(10 citation statements)

References 27 publications

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“…A mutagenic effect was observed with Vent (exo -) DNA polymerase in the presence of 7-8 % (v/v) propan-1-ol, achieving error rate up to 9.8 x 10 -3 mutation/bp/PCR. This bias differs interestingly from Mn 2+ -mediated epPCR that favors substitution of As and Ts (75 % A/T N, 25 % G/C N) [82]. This bias differs interestingly from Mn 2+ -mediated epPCR that favors substitution of As and Ts (75 % A/T N, 25 % G/C N) [82].…”

Section: Heavy Metal and Organic Solvent-mediated Eppcr Methodsmentioning

confidence: 84%

“…The influence of urea, isopropanol, propan-1-ol, and butan-1-ol on PCR fidelity and yield was investigated for three DNA polymerases by Beauregard and coworkers [82]. A mutagenic effect was observed with Vent (exo -) DNA polymerase in the presence of 7-8 % (v/v) propan-1-ol, achieving error rate up to 9.8 x 10 -3 mutation/bp/PCR.…”

Section: Heavy Metal and Organic Solvent-mediated Eppcr Methodsmentioning

confidence: 99%

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The Diversity Challenge in Directed Protein Evolution

Wong¹,

Zhurina²,

Schwaneberg

2006

CCHTS

100

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Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships. This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and cost-effectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle). We hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.

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Section: Heavy Metal and Organic Solvent-mediated Eppcr Methodsmentioning

confidence: 84%

Section: Heavy Metal and Organic Solvent-mediated Eppcr Methodsmentioning

confidence: 99%

The Diversity Challenge in Directed Protein Evolution

Wong¹,

Zhurina²,

Schwaneberg

2006

CCHTS

100

View full text Add to dashboard Cite

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“…Apart from this, several protocols have been developed with the aim of increasing the error rate of Taq polymerase, which can infinitely be varied by increasing the concentration of MgCl 2 , 153 addition of MnCl 2 , 15 using unbalanced concentrations of nucleotides, 16 using a mixture of triphosphate nucleoside analogs, 146 or a combination of all these to achieve higher rates of mutations. Similarly alcohol mediated epPCR is another method reported by Claveau et al 24 to increase the error rates. In the presence of urea, isopropanol, propan-1-ol, butan-1-ol polymerases were active until only a critical concentration, beyond which they were progressively inhibited.…”

Section: Error Prone Pcr (Eppcr)mentioning

confidence: 96%

Directed Evolution: An Approach to Engineer Enzymes

Kaur

Sharma

2006

Critical Reviews in Biotechnology

127

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Directed evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

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“…Critical concentrations of alcohols generally increased with the thermoresistance of the polymerases and decreased their activity. Comparison of various PCR conditions indicates that ethanol has specific mode of action by partial destabilization of the polymerase [15]. Lower concentrations of EDTA produced varying degrees of inhibition [12].…”

Section: Bsa (67 Kd)mentioning

confidence: 99%

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

Popovski

Nestorovski

Svetozarevic

et al. 2017

Maced. J. Chem. Chem. Eng.

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Тhermophilic bacteria Bacillus caldolyticus isolated from the hot spring in Bansko, Republic of Macedonia, were used for the isolation of DNA polymerase. Bacterial cells were disrupted by sonication and the first step in the purification of DNA polymerase was 40% ammonium sulfate precipitation. This was followed by chromatographic procedures on Sephadex G-50, DE-52 and CM-52 cellulose. DNA polymerase activity was analyzed at each step of purification using the incorporation of 3 H dATP in activated calf thymus DNA. The purity of the DNA polymerase was analyzed on SDS PAGE. Optimal conditions of activity were determined for temperature, pH, dNTP and Mg ++ concentration. In addition, the effect of ethanol and EDTA as possible inhibitors of polymerase activity was also analyzed. The optimal temperature of DNA polymerase was 66 ºC; the optimal pH was 7.2, optimal MgCl 2 concentration was 2.5 mM, and the optimal substrate concentration was 2.5 × 10 -6 M dNTP. The inhibitory effect of EDTA and ethanol on DNA polymerase was above 10 mM and 10%, respectively. Keywords: Bacillus caldolyticus; DNA polymerase; purification; optimization; inhibitors ПРОЧИСТУВАЊЕ И ОПТИМИЗИРАЊЕ НА УСЛОВИТЕ ЗА ДНК-ПОЛИМЕРАЗА ИЗОЛИРАНА ОД ТЕРМОФИЛНАТА БАКТЕРИЈА BACILLUS CALDOLYTICUSТермофилната бактерија Bacillus caldolyticus изолирана од термалниот извор во Банско, Република Македонија, беше користена за изолација на ДНК-полимераза. Бактериските клетки се лизирани со ултразвук и првиот чекор во прочистувањето на ДНК-полимеразата беше исталожување со 40% амониум сулфат. Потоа следуваа хроматографски процедури на Sephadex G-50, DE-52 и CM-52 целулоза. Активноста на ДНК-полимеразата беше анализирана во секој степен на прочистувањето со инкорпорација на 3 H dATP во активирана ДНК од телешки тимус. Чистотата на ДНК-полимеразата беше анализирана со користење на SDS PAGE. Оптималните услови за активноста беа одредени за температура, pH, dNTP и Mg ++ . Дополнително беа анализирни ефектите на етанолот и EDTA како можни инхибитори на ДНК-полимеразната активност. Оптималната температура за оваа ДНК-полимераза е 66 ºC, оптималната pH е 7.2, оптималната концентрација на MgCl 2 е 2.5 mM, додека оптималната концентрација на dNTP е 2.5 × 10 -6 M. EDTA покажува инхибиторен ефект врз ДНК-полимеразата во концентрација над 10 mM, додека етанолот во концентрација над 10%.

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Alcohol-Mediated Error-Prone PCR

Cited by 13 publications

References 27 publications

The Diversity Challenge in Directed Protein Evolution

The Diversity Challenge in Directed Protein Evolution

Directed Evolution: An Approach to Engineer Enzymes

Purification and optimization of conditions for DNA polymerase isolated from thermophile bacteria Bacillus caldolyticus

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