应用聚合链锁反应技术与限制切割多型性检测紧迫敏感猪.PDF

應用聚合 鏈鎖反應技術與限制 切割多型性檢測緊迫敏 感豬 陳銘正 林佳靜 李盈漢吳伊倫 李易儒 林瑞毓張俊達 王祥宇 國立宜蘭技術學院 畜產系 摘要 本試驗之目的乃建立聚合 鏈鎖反應(polymerase chain reaction , PCR) 與限制 切割多型性(restriction fragment length polymorphism, RFLP)技 術，檢測豬鈣離子釋放管道 (calcium release channel , CRC)基因是否突變， 俾快速鑑定豬緊迫敏感徵候群 (porcine stress syndrome , PSS)基因。豬血液 與精液樣品萃取之基因組DNA(genomic DNA, gDNA)做為 PCR之模板，以 CRC 基 因之 1614-1633鹼基序列為上游引子，另以 1957-1976鹼基序列之互補序列為下 游引子，經聚合 鏈鎖反應後得 363鹼基對 (base pair, bp)之 DNA片段。以 Hha I限制 切割此片段，經電泳後可由限制 分切片段區分緊迫敏感基因為純合子 或雜合子。正常純合子、雜合子、PSS 純合子三者之DNA片段長度組合分別為(231, 132 bp) 、(363, 231, 132 bp)與(363 bp) 。採樣分析宜蘭某豬場豬隻CRC遺傳 型，發現正常純合子、雜合子與 PSS 純合子之豬隻分別佔60 % 、35 %與5 % ，估 計緊迫敏感基因頻率為 22.5 % 。應用PCR技術可以快速判定 PSS豬隻，且可準 確分辨基因型為雜合子或純合子。 關鍵詞：聚合 鏈鎖反應、鈣離子釋放管道、豬緊迫敏感徵候群 The Use of Polymerase Chain Reaction and Restriction Fragment Length Polymorphism to Detect the Gene for Porcine Stress Syndrome Ming-Cheng Chen Chai-Ching Lin Yieng-Han Lee Yi- Luen Wu Yi-Ru Lee Ruei-Yu Lin Chiun-Da Chan Hsiang-Yu Wang Department of Animal Science National Ilan Institute of Technology Abstract Pigs with mutated CRC gene have genetic disorder which can result in porcine stress syndrome (PSS). The purpose of this study was to establish polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) techniques for detecting mutation of calcium release channel (CRC) gene among pigs. All genomic DNA extracted from pig blood and semen samples were provided as PCR templates. The forward primer used in PCR amplification was corresponding to nucleotides 1614 to 1633, whereas the reverse primer was complementary to nucleotides 1957 to 1976 of CRC gene. A 363-bp nucleotide fragment was a