利用引子延伸反應於微珠微流道系統進行單核苷酸多態性分型

Abstract

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。這種變異為一重要的生物分子遺傳標記，其影響了物種個體間性狀的差異，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等位基因特異性引子延伸(primer extension)反應技術，並整合微珠微流道(bead-based microfluidics)技術以減少試劑使用量、改善反應動力學、增強訊號強度。本系統採用微珠作為固態載體，將參與聚合酶連鎖反應之正向引子固定於微珠表面，而後利用反向引子於微流道系統中進行等位基因特異性引子延伸反應，以進行擴增曲線分析達成單核苷酸多型性之基因分型。本研究中以白蛋白突變基因(Albumin mutated, ALB)為例，成功以整合了聚合酶連鎖反應與單核苷酸多型性檢測程序之微珠微流道系統完成台灣土雞取得之基因體DNA位點ALB之基因分型。

Single nucleotide polymorphism (SNP), a single nucleotide variation occurring in sequence of deoxyribonucleic acid (DNA), can serve a crucial bio-marker affecting individuals’ phenotypes and draw great attention in medical, agriculture and breeding fields. Most of the SNP genotyping techniques today utilize enzymes or modification of DNA, leading to the requirement of high reagent cost and a series of complex procedures. Herein, we present our recent effort to integrate the SNP genotyping procedures onto a microchip by adapting a primer extension technique onto microbeads, which permit better hybridization kinetics and higher signal-to-noise ratio. The primer extension is based on allele-specific quantitative polymerase chain reaction (AS-qPCR) techniques for SNP allelic discrimination by the capability of the reverse primer extended with nucleotides during thermal cycles. In addition, our bead-based microfluidic chip can provide a rapid thermal response, which minimize the non-specific reactions, thus greatly improve the specificity. With only six thermal cycles, our proposed device successfully distinguishes the SNP genotypes of Albumin (ALB) from genomic DNA of Taiwan country chicken. The results show a much quicker detection while the DNA amplification and detection procedures are integrated for Lab-on-a-Chip applications.