本研究是以allele-specific polymerase chain reaction (AS-PCR)方法來進行hMYH基因中單核?酸多態性(single nucleotide polymorphism，簡稱SNP)分析。首先以質體DNA(含hMYH基因)作為模板，建立AS-PCR的最佳檢測條件。先前﹐我們已就AS-PCR方法﹐探討了DNA濃度?PCR黏貼溫度?dNTP濃度等因素對AS-PCR靈敏度及準確性的影響。本研究繼續對AS-PCR方法作進一步的改善﹐我們對引子3’’ 端作了不同的修飾﹐以尋求更好的AS-PCR條件。同時﹐我們對非小細胞肺癌(NSCLC)細胞作了初步的研究﹐研究結果並未顯示所選擇的NSCLC細胞具有hMYH基因上的突變熱點G382D、Y165C及V232F﹐但發現NSCLC細胞在hMYH基因的exon15~16部份具有不同尋常的序列﹐分析結果顯示其包含異常的Alu系列結構。由于Alu系列在人類基因體中的移動與人類多種疾病﹐包括癌症﹐有非常大的關係﹐故研究結果暗示了hMYH基因中Alu系列的異常可能影響hMYH修復的活性﹐從而與非小細胞肺癌的形成有一定的關係。 此結果也為我們未來的研究提供了一定的方向。 In this study, we employed modified allele-specific polymerase chain reaction (AS-PCR) to analysis the mutations in hMYH. At first, we utilized the plasmid DNA containing hMYH as the DNA template to establish the optimum AS-PCR conditions. Previously, we have investigated the effects of DNA template concentration, annealing temperature, as well as imbalanced dNTP on the reliability and sensitivity of AS-PCR. Here, we further explored the effect of various modifications at the 3’’ terminal of the primers on the efficiency of AS-PCR. We also applied the established AS-PCR condition to examine the hMYH mutations in human non-small cell lung cancer (NSCLC) cells. Our preliminary results indicated no obvious mutations of hMYH at G382D, Y165C, and V232F hot-spots. However, we found that the sequence between exon 15~16 exhibited different sequence structures from that published by GeneBank, which was identified as the Alu repeated sequence. Since the Alu sequences has also been implicated in several inherited human diseases, including various forms of cancer. Our results may suggest the possible connection between the abnormal Alu sequence and the lost of hMYH activity, in which leads to the cell transformation and cancer predisposition. Therefore, the preliminary results bring us a new direction on the study of the correlation between hMYH and the NSCLC formation.
