建立AS-PCR分析hMYH基因多態性之方法及其於大腸直腸癌之檢測應用

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Title:	建立AS-PCR分析hMYH基因多態性之方法及其於大腸直腸癌之檢測應用
Other Titles:	Detection of the SNPs of hMYH in colorectal cancer by modified AS-PCR methods.
Authors:	王心潔 Wang,Shin-Jie
Contributors:	顧野松 Gu, Ye-Song 東海大學化學工程與材料工程學系
Keywords:	硫代磷酸化;大腸直腸癌;單核?酸多態性;AS-PCR;鎖核酸 colorectal cancer;single nucleotide polymorphism (SNP);allele-specific polymerase chain reaction (AS-PCR);phosphorothioate modification;locked nucleic acid modification
Date:	2011
Issue Date:	2011-10-12T07:52:34Z (UTC)
Abstract:	研究發現，人類hMYH雙等位基因突變(biallelic germline mutations，G：C→T︰A)與大腸直腸癌及其他癌症的產生有密切的關係。因此如何建立一套「早期發現，早期治療」的完善檢測分析是現今最重要的課題。本研究是以Allele Specific-PCR (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，並進一步針對如何提高AS-PCR靈敏度進行探討。由實驗結果發現，對偶性引子與DNA模板間之錯配模式設計為嘌呤︰嘌呤配對，亦或嘧啶︰嘧啶配對時，所呈現之ON/OFF訊號較清楚易分辨。另外，也證明了在對偶性引子的3’端連續設計兩個鹼基錯配，可有效的提高AS-PCR之靈敏度。此外，我們也同步建立其他新的檢測方法–ON/OFF switch assay與OFF/ON switch assay檢測hMYH基因之突變熱點–Y165C，企圖提升檢測hMYH基因中SNPs分析之可信度。由ON/OFF switch assay之實驗結果得知，硫代磷酸化修飾(phosphorothioate-modified)之特異性引子搭配Pfu polymerase於適當之黏合溫度時，所呈現之ON/OFF訊號較明確；而鎖核酸修飾(locked nucleic acid modification，簡稱LNA modification)則與Taq polymerase一同進行PCR之分析訊號較佳。 The molecular biology of cancer literatures have shown that colorectal cancers are associated with biallelic germline mutations of the human MutY homologue gene (hMYH).Therefore, how to establish a perfect detection mothod for detecting hMYH mutations to achieve “early diagnosis and early treatment” is the one of the most important issues nowadays.In this study, we have detected single nucleotide polymorphism (SNP) genotyping by using allele-specific polymerase chain reaction (AS-PCR), and also disscused how to improve the capability ability for SNPs of hMYH by AS-PCR. The experimental results have shown that it is clearly to distinguish with the ON/OFF singals for mismatch between the allele specific primer and template by the purine-purine and pyrimidine-pyrimidine mismatching model.In addition, we also established other new methods such as ON/OFF switch assay and OFF/ON switch assay for the analysis of one of hMYH mutation, Y165C, regard to colorectal cancer. We attempted to enhance the reliability of the AS-PCR for hMYH gene analysis. Our results indicated that ON/OFF method displayed expected on/off signal to distinguish the mismatch at phosphorothioate-modified primers with pfu polymerase under a suitable annealing temperature. Meanwhile, the ON/OFF method was successful by using locked nucleic acid-modified primers with Taq polymerase under established conditions.
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