- 學位論文
質譜儀應用於DNA聚合酶校正活性分析
DNA Polymerase Proofreading Assay by MALDI-TOF Mass Spectrometry
摘要
核酸聚合酶在生物體參與許多重要的功能，包括核酸複製、核酸修復、基因重組、反轉錄，以及維持遺傳的恒定。核酸聚合酶的校正功能在避免突變上非常重要。核酸聚合酶的忠誠度依靠在引子末端加入正確的核苷酸，同時將意外裝錯的核苷酸移除。 研究核酸聚合酶校正活性的方法包括放射性標定後凝膠電泳分析。目前之方法通常費時， 耗人工且必須關注輻射安全。 發光方式的測定也被建立， 使用核苷類似物2甲基嘌呤的內在螢光標定，曾大量應用於聚合酶校正分析， 但這個方法受限於2-AP可能與C形成熱動力穩定的配對，另2-AP:T也會被某些聚合酶選擇性排斥。 MALDI-TOF 質譜儀的高解析能力被發展成檢測DNA 的 PinPoint assay，即利用四種ddNTPs進行引子延伸反應，再以質譜儀分析產物，以鑑別在特定基因位置的多形性。我們發現這個方法可以修改成研究核酸聚合酶校正能力的很好工具。 我們設計出模仿引子模板交界的寡核酸，在引子3'端與模板形成12 種可能的配對錯誤, 再用大腸菌核酸聚合酶I(PolI)進行校正反應，從質譜儀的結果，呈現出12種配對錯誤皆可被PolI校正，比起其它非質譜方法，質譜儀測定法快同時也省人力。質譜結果也可提供有關反應機制的訊息。這個質譜儀分析校正活性的方法，也成功地呈現T4與T7 核酸聚合酶的校正活性。
並列摘要
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.