| Summary: | 碩士 === 國立臺灣大學 === 醫學檢驗暨生物技術學研究所 === 104 === DNA polymerases play an important role in various cellular processes including DNA replication, DNA repair, genetic recombination, reverse transcription and maintenance of genomic stability. The proofreading function of DNA polymerase is critical in preventing mutations. The fidelity of DNA polymerases depends on its ability to incorporate the appropriate nucleotides into the growing DNA strand and removing mis-incorporated nucleotide at template-primer junction.
Traditional methods for the detection of DNA polymerases proofreading ability include gel-based assays and radioisotopic labeling. However, these methods are generally time-consuming and labor-intensive and require necessary safety measures to control
radioactive exposure. Luminescent-based methods for the detection of polymerase proofreading activity were also developed by using intrinsically fluorescent nucleotide analogs , such as 2-aminopurine (2-AP) which has been extensively employed to study polymerase proofreading activity in vitro. However, these methods suffer from low specificity due to the ability of 2-AP to form thermodynamically stable base pairs with cytosine and 2-AP formed non-canonical base pair is subject to selective discrimination by some DNA polymerases.
The high resolution of MALDI-TOF (matrix-assisted laser desorption ionization mass spectrometry with time-of-flight) for DNA detection has been employed in the PinPoint assay in which the primer extension reactions with four unlabeled ddNPTs followed by an analysis by MALDI-TOF identify the polymorphism at a given locus. We suppose the concept of this approach can be modified to study DNA polymerase proofreading.
We employed double strand synthetic oligonucleotides with all 12 possible terminal mismatches at the template-primer junction to mimic mis-incorporated nucleotide as proofreading substrates for E. coli DNA polymerase I (Pol I). From MALDI-TOF analysis, we demonstrate that all 12 mismatches can be actively corrected by Pol I. This method is faster and less laborious than non-mass spectrophotometric methods. The results in mass spectrum also provide additional information for reaction mechanism. The mass spectroscopy analysis of proofreading was also tested for other DNA polymerases such as T4 and T7 DNA polymerases with success.
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