以BAMPER進行單一核苷酸多型性之分析

Analysis of Single Nucleotide Polymorphism by BAMPER

簡郁沛(Yu-Pei Chien)

指導教授 ： 游吉陽

大同大學/工程學院/生物工程學系所/碩士(2006年)

摘要

單一核苷酸多型性（Single Nucleotide Polymorphism，SNP）為遺傳上最常見的基因序列變異。部分SNP與癌症、藥物代謝、遺傳疾病等有關。目前應用在分析SNP之技術包括DNA微陣列、pyrosequencing、Sanger基因定序等。
本研究係以BAMPER（Bioluminometric Assay Coupled With Modified Primer Extension Reactions）為基礎。當引子序列與含有SNP之模板完全互補時，DNA聚合酶可進行延伸反應。每加入一個dNTP分子，即產生一分子的PPi。PPi經ATP sulfurylase 與firefly luciferase催化，可發出波長562 nm之生物冷光。若模板無法與引子完全互補，則產生之冷光強度會大幅降低，藉由冷光強弱之分析，可進行基因型之判別。
以含有D-amino acid oxidase基因之pET-23a (＋)質體為分析樣本。使用artificial mismatch primer（AMP），當基因型為heterozygous時，其冷光強度比為1.0 ± 0.07 (n = 3)，homozygous之冷光強度比可達5.6 ± 1.8（mutant，n = 3）及4.5 ± 1.1（wild type，n = 3）。考慮3倍之標準偏差，則冷光ratio達1.21以上即可判定為homozygous。Allele specific primer extension反應前藉pyrophosphatase清除背景PPi，對後續冷光分析則無明顯幫助。
試以酵素固定化的方式進行本實驗，以期結合微流體原件發展新型SNP檢測晶片。酵素固定化使用Immunodyne® ABC Membrane做為載體，以三組不同之酵素反應濃度進行試驗。酵素固定化時若以0.71 mg/mL之firefly luciferase與0.14 mg/mL之ATP sulfurylase進行，對SNP基因型判別有最佳之效果。

關鍵字

單一核苷酸多型性

並列摘要

Single nucleotide polymorphism ( SNP ) is the most frequent type of genetic variation in human. Certain SNPs are related to cancers, drug metabolism, and genetic disorders. A wide variety of techniques have been developed to analyze SNPs, including DNA microarray, pyrosequencing, and Sanger sequencing.
This manuscript is based on upon Bioluminometric Assay Coupled With Modified Primer Extension Reactions ( BAMPER ). When a specifically designed primer is fully complementary to the template containing SNP site, the DNA polymerase can incorporate the deoxynucleotides to extend the primer. One PPi molecule is released whenever a deoxynucleotide is attached to the growing primer. PPi is further converted to green-yellow bioluminescence (562 nm) by ATP sulfurylase and firefly luciferase. However, when the primer is not fully complementary to the template, the bioluminescence intensity is greatly decreased. The SNP genotype can be identified by comparing bioluminescence intensity when a pair of SNP primers are used.
A plasmid（pET-23a (＋)）containing D-amino acid oxidase gene was used as model analyte. When artificial mismatch primer（AMP）was applied, the bioluminescence ratio was 1.0 ± 0.07 (n=3) for heterozygous genotype. For homozygous genotypes, the ratios were 5.6 ± 1.8 (mutant, n = 3) and 4.5 ± 1.1 (wild type, n = 3). Considering three times of the standard deviation of the heterozygous, result with bioluminescence ratio greater than 1.21 can be called as homozygous. We also attempted to clean up the background PPi in the deoxynucleotides, nonetheless, the background removal did not facilitate the SNP identification.
In order to develop microfluidic SNP analysis device, we tested the possibility of using immobilized enzymes in BAMPER. The Immunodyne® ABC Membrane was selected as support and three different combinations of enzyme concentrations were tested. 0.71 mg/mL firefly luciferase and 0.14 mg/mL ATP sulfurylase were found optimal.

並列關鍵字

SNP

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