Detection of Cellular DNA Cleavage Using Non-Proofreading Thermostable DNA Polymerases
نویسندگان
چکیده
منابع مشابه
Detection of cellular DNA cleavage using non-proofreading thermostable DNA polymerases.
Few techniques are available to detect DNA lesions in cultured cells at the nucleotide level (8). One such method is primer extension of genomic DNA (5) that may be improved using linear amplification by repeated PCR cycles (1,11). This method has been used successfully to map the genomic sites of topoisomerase II (2,3,7) and topoisomerase I activity (10). DNA topoisomerases modulate DNA struct...
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DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequen...
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The amplification efficiencies of several polymerase chain reaction (PCR) enzymes were compared using real-time quantitative PCR with SYBR Green I detection. Amplification data collected during the exponential phase of PCR are highly reproducible, and PCR enzyme performance comparisons based upon efficiency measurements are considerably more accurate than those based on endpoint analysis. DNA p...
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Kinetic theory and thermodynamics are applied to DNA polymerases with exonuclease activity, taking into account the dependence of the rates on the previously incorporated nucleotide. The replication fidelity is shown to increase significantly thanks to this dependence at the basis of the mechanism of exonuclease proofreading. In particular, this dependence can provide up to a 100-fold lowering ...
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ژورنال
عنوان ژورنال: BioTechniques
سال: 2000
ISSN: 0736-6205,1940-9818
DOI: 10.2144/00286bm01