利用微珠與微流道溫度梯度場分析熔解曲線進行單核苷酸多態性檢測

Bead-based Melting Analysis in Temperature-gradient Microchannel for Single Nucleotide Polymorphism Detection

李侃謙(Kan-Chien Li)

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(2013年)

https://doi.org/10.6342/NTU.2013.10594

摘要

單核苷酸多型性 (Single Nucleotide Polymorphism, SNP)為去氧核醣核酸(DNA)的序列中發生一個鹼基對的變異。這類的變異若發生在編碼區則有可能引起蛋白質氨基酸編碼改變進而影響原本正常功能的表現。檢測這類的基因變異，去氧核醣核酸熔解曲線(DNA熔解曲線)的檢測方式是一個非常經濟且有效的手段。近年來，許多研究將微流體平台大量應用於熔解曲線的分析，主因於其技術不但可以大幅減少使用試劑，並且能微小化檢測平台，成為重點照護的檢測裝置。目前以微流體檢測DNA熔解曲線研究中，為了避免檢體在前處理及檢測時交叉污染，只能以單次檢測單項、固定同一檢體於個別區域等方式進行低通量的檢測。為了改善這些限制，本研究以毛細血管擴張性運動失調突變基因(ataxia-telangiectasia mutated, ATM) 為例，將個別檢體固定於不同群體的微珠上，並且循序注入內建微溫度場的微流道中進行基因檢測，成功由藍瑞斯母豬取得之基因體DNA完成位點ATM-A的基因分型。

關鍵字

微珠 ； 微流道 ； 單核苷酸多型性 ； 氧核醣核酸熔解曲線分析 ； 毛細血管擴張性運動失調突變基因

並列摘要

Deoxyribonucleic acid (DNA) melting analysis is a powerful tool in genotyping and mutation scanning. The recent development in microfluidics further promotes melting analysis within a miniaturized device, which not only can reduce the reagent cost but also provide the possibility to perform point-of-care molecular diagnosis. The DNA melting analysis in microfluidic devices usually resorts to solid or liquid phase on sample preparations, in which either DNA immobilization on channel surfaces is required or only single analysis is allowed. To address these limitations, a bead-based melting analysis in continuous flow configuration is proposed. Our device is tested on detecting single nucleotide polymorphisms (SNPs) - one of the most important DNA sequence variations for human, animals and other species. The samples with ataxia telangiectasia-mutated (ATM) genes from Landrace sows, which play important roles in total number of piglets born, number born alive and average birth weight due to its differential expression between the morula and blastocyst stages, were chosen as our SNP genotyping candidates.

並列關鍵字

microfluidics ； single nucleotide polymorphisms ； ataxia telangiectasia-mutated ； bead-based melting analysis

參考文獻

Bortolin, S., M. Black, H. Modi, I. Boszko, D. Kobler, D. Fieldhouse, E. Lopes, J. M. Lacroix, R. Grimwood, P. Wells, R. Janeczko and R. Zastawny. 2004. Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: Application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms. Clinical Chemistry 50:2028-2036.

Brookes, A. J. 1999. The essence of SNPs. Gene 234:177-186.

Chung, T. D. and H. C. Kim. 2007. Recent advances in miniaturized microfluidic flow cytometry for clinical use. Electrophoresis 28:4511-4520.

Crews, N., C. T. Wittwer, J. Montgomery, R. Pryor and B. Gale. 2009. Spatial DNA Melting Analysis for Genotyping and Variant Scanning. Analytical Chemistry 81:2053-2058.

Deng, T., G. M. Whitesides, M. Radhakrishnan, G. Zabow and M. Prentiss. 2001. Manipulation of magnetic microbeads in suspension using micromagnetic systems fabricated with soft lithography. Applied physics letters 78:1775-1777.