應用微珠微流道及等位基因特異性雜交技術於單核苷酸多型性檢測

Single Nucleotide Polymorphism Detection Using DASH Technology in Bead-Based Microfluidics

高培鈞(Pei-Chun Kao)

指導教授 ： 盧彥文

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.01688

摘要

單核苷酸多型性 (Single Nucleotide Polymorphism)，為最常見之基因變異，這種變異亦為一重要的生物分子遺傳標記，其不但影響了物種個體間之差異，亦可能造成個體具有過敏體質、遺傳性疾病或是對於藥物之抗藥性等。目前多數的單核苷酸多型性檢測技術仍存在缺點，如須要耗費大量的試劑以及繁複之處理過程。因此，本研究致力於發展微小化之動態等位基因特異性雜交法技術，並整合微珠微流道技術以減少試劑使用量。本系統採用微珠作為固態載體，將待測的去氧核醣核苷酸固定於微珠表面，而後利用流體將微珠限制於流道固定位置，以進行熔解曲線分析達成單核苷酸多型性之基因分型。本研究中以ataxia-telangiectasia突變 (ATM) 基因為例，成功以合成DNA以及由藍瑞斯母豬取得之基因體DNA完成位點ATM-A之基因分型。此微珠微流道系統成功地減少試劑使用量，並簡化了聚合酶鏈鎖反應之程序，同時保有了動態等位基因特異性雜交法之靈活性及準確度。

關鍵字

微珠微流道 ； 單核苷酸多型性 ； 熔解曲線分析 ； 動態等位基因特異性雜交法 ； ataxia-telangiectasia突變基因

並列摘要

Single-nucleotide polymorphism (SNP) not only is one of the most common genetic variances in human genome, but also serves a crucial bio-marker greatly affecting phenotypes of individuals. Most of today’s SNP detection techniques utilize enzymes or modification of DNA, leading to the requirement of high reagent cost or complex procedures. Thus, a SNP genotyping system is developed by integrating a miniaturized Dynamic Allele-Specific Hybridization (DASH) technique with a bead-based microfluidic device. The microbeads function as a solid vehicle for immobilizing DNA duplexes, confined by the fluidic traps in the device. Meanwhile, the Melting Curve analysis of the DASH technique was applied for SNP detection. Our proposed device was successfully tested, detecting the SNP of ataxia-telangiectasia-mutated (ATM-A) from both synthetic DNA and genomic DNA of Landrace sows. This bead-based device required a minimal reagent amount and simplified Polymerase Chain Reaction (PCR) procedures in preparations, while preserved as a flexible and accurate SNP detection scheme by integrating with DASH technique.

並列關鍵字

bead-based microfluidc ； single nucleotide polymorphism ； dynamic allele-specific hybridization ； melting curve analysis ； ataxia-telangiectasia mutated gene

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