Evolving a Thermostable DNA Polymerase That Amplifies from Highly Damaged Templates

Gloeckner, Christian; Sauter, Katharina B. M.; Marx, Andreas

doi:10.1002/anie.200603987

Cited by 49 publications

(41 citation statements)

References 37 publications

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“…A 1657 base pairs DNA fragment was amplified by PCR and cloned into a suitable expression vector, transformed into E. coli cells and several single clones analysed by sequencing. With an error rate of 2.8×10 −5 mutations per base per duplication KlenTaq R660V exhibits somewhat improved fidelity than KlenTaq wild-type with 8.8×10 −5 mutations per base per division (Table 3) [63]. Notably, mainly the number of transitions dropped whereas the number of transversions is unaffected.…”

Section: Resultsmentioning

confidence: 99%

“…aNumber of mutations per 650 bases ( KlenTaq wild-type) or 726 bases (R660V) sequenced per clone.bError rate equals number of mutations per base per division.cData taken from [63].…”

Section: Resultsmentioning

confidence: 99%

“…KlenTaq and its respective mutants were recombinantly expressed in E. coli BL21 (DE3) and purified with Ni-IDA as previously described[63]. Enzyme purity and quantity were determined by SDS-PAGE using an albumin standard dilution curve.…”

Section: Methodsmentioning

confidence: 99%

“…PCR reactions (100 µl) contained 30 fmol template (pGDR11-codon optimized gene encoding KlenTaq wild-type[63]), 250 µM of each dNTP, forward primer [400 nM, 40-nt, 5′-d(GGA TCC GCA TGC AGC ACT GGA AGA AGC ACC TTG GCC TCC G)-3′] and reverse pimer [400 nM, 32 nt, 5′-d(CTA ATT AAG CTT TTA TTC TTT TGC AGA CAG CC)-3′], 80 nM of DNA polymerase in 50 mM Tris-HCl (pH 9.2), 16 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 2.5 mM MgCl 2 . After an initial denaturation cycle (95°C for 1 min), the product was amplified by 30 PCR cycles (95°C for 60 s, 65.1°C for 60 s and 72°C for 2 min).…”

Section: Methodsmentioning

confidence: 99%

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Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

et al. 2014

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The selectivity of DNA polymerases is crucial for many applications. For example, high discrimination between the extension of matched versus mismatched primer termini is desired for the detection of a single nucleotide variation at a particular locus within the genome. Here we describe the generation of thermostable mutants of the large fragment of Thermus aquaticus DNA polymerase (KlenTaq) with increased mismatch extension selectivity. In contrast to previously reported much less active KlenTaq mutants with mismatch discrimination abilities, many of the herein discovered mutants show conserved wild-type-like high activities. We demonstrate for one mutant containing the single amino acid exchange R660V the suitability for application in allele-specific amplifications directly from whole blood without prior sample purification. Also the suitability of the mutant for methylation specific amplification in the diagnostics of 5-methyl cytosines is demonstrated. Furthermore, the identified mutant supersedes other commercially available enzymes in human leukocyte antigen (HLA) analysis by sequence-specific primed polymerase chain reactions (PCRs).

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Section: Resultsmentioning

confidence: 99%

“…aNumber of mutations per 650 bases ( KlenTaq wild-type) or 726 bases (R660V) sequenced per clone.bError rate equals number of mutations per base per division.cData taken from [63].…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

et al. 2014

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“…The DNA polymerase KTqM747K is a mutant of the N-terminally truncated A-family Taq polymerase. It is thermostable and can efficiently bypass various DNA lesions (25,32,33). We found that the O 6 -alkylG adducts template the specific incorporation of the artificial nucleotide Benzi MP when DNA synthesis was carried out by KTqM747K .…”

Section: Introductionmentioning

confidence: 99%

The use of an artificial nucleotide for polymerase-based recognition of carcinogenicO⁶-alkylguanine DNA adducts

et al. 2016

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Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O6-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O6-methylguanine (O6-MeG) and O6-carboxymethylguanine (O6-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2′-deoxynucleoside-5′-O-triphosphates (BenziTP and BIMTP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide Benzi opposite adducts, with up to 150-fold higher catalytic efficiency for O6-MeG over guanine in the template. Furthermore, addition of artificial nucleotide Benzi was required for full-length DNA synthesis during bypass of O6-CMG. Selective incorporation of the artificial nucleotide opposite an O6-alkylguanine DNA adduct was verified using a novel 2′,3′-dideoxy derivative of BenziTP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BenziTP opposite biologically relevant O6-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies.

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Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries

Gloeckner

Kranaster

Marx

2010

CP Chemical Biology

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The protocols in this article describe the construction of a mutant DNA polymerase library using error‐prone PCR (epPCR) as a method for gene randomization, followed by screening of the library using two different approaches. The examples described use an N‐terminally truncated form of the thermostable DNA polymerase I of Thermus aquaticus (Taq DNA polymerase), namely Klentaq (KTQ), and protocols are included for the identification of variants with (1) increased DNA lesion‐bypass ability and (2) enhanced selectivity for DNA match/mismatch recognition. The screening assays are based on double‐stranded DNA detection (using SYBR Green I) which can be carried out using standard laboratory equipment. The described assays are designed for use in a 384‐well plate format to increase screening throughput and reduce material costs. For improved accuracy and ease of liquid handling, the use of an automated liquid handling device is recommended. Curr. Protoc. Chem Biol. 2:89‐109. © 2010 by John Wiley & Sons, Inc.

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Evolving a Thermostable DNA Polymerase That Amplifies from Highly Damaged Templates

Cited by 49 publications

References 37 publications

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

The use of an artificial nucleotide for polymerase-based recognition of carcinogenicO⁶-alkylguanine DNA adducts

Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries

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Evolving a Thermostable DNA Polymerase That Amplifies from Highly Damaged Templates

Cited by 49 publications

References 37 publications

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

Variants of a Thermus aquaticus DNA Polymerase with Increased Selectivity for Applications in Allele- and Methylation-Specific Amplification

The use of an artificial nucleotide for polymerase-based recognition of carcinogenicO6-alkylguanine DNA adducts

Directed Evolution of DNA Polymerases: Construction and Screening of DNA Polymerase Mutant Libraries

Contact Info

Product

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The use of an artificial nucleotide for polymerase-based recognition of carcinogenicO⁶-alkylguanine DNA adducts