- 學位論文
企鵝目鳥類性別高通量鑑定方法
High-throughput avian gender identification of Order Sphenisciformes
摘要
CHD（染色體DNA解旋酶結合蛋白）特異性PCR（聚合酶連鎖反應）和膠體電泳是最常見的鳥類分子性別鑑定技術，但這是一種很密集的使用PCR與凝膠來進行實驗的方法。 性別鑑定的失敗往往與CHD- Z和CHD-W這兩個基因的PCR產物長度差異太短，這樣沒辦法快速達成高通量鳥類性別的分子生物鑑定。 我的研究的目的是設計引子或者探針去高通量性別鑑定的企鵝目的物種。 我將介紹一個 PCR 熔點曲線分析（Melting Curve Assays, MCA）來鑑定鳥類的性別，這是一個不用使用凝膠並且快速又便宜的鑑定方法。 目前，我主要重點放在高通量的巴布亞企鵝性別鑑定(Pygoscelis papua)。 使用Griffiths的P2/P8作為引子進行PCR與膠體電泳的實驗，並且進行PCR/MCA實驗都沒辦法正確判定巴布亞企鵝的性別。在進行DNA的轉型與轉殖、定序後，針對巴布亞企鵝的CHD-Z和CHD-W特異性重新設計了引子，並且進行了 PCR /MCA實驗。使用此特異性引子，針對10隻巴布亞企鵝同時進行了鑑定，並且利用CHD-W與CHD-Z的PCR產物熔點不同而加以區別。 因此，高通量的鳥類分子性別鑑定技術被成功地應用於巴布亞企鵝。
並列摘要
Combination of CHD (chromo-helicase-DNA binding protein)-specific polymerase chain reaction (PCR) with electrophoresis (PCR/electrophoresis) is the most common avian molecular sexing technique but it is lab-intensive and gel-required. Gender determination often fails when the difference in length between the PCR products of CHD-Z and CHD-W genes is too short to be resolved and it is less high-throughtput. The purpose of my study is to design the primers or probes for the high-throughput gender identification of several species for the Order Sphenisciformes (penguins). I will introduce a PCR-melting curve analysis (PCR/MCA) to identify the gender of birds by genomic DNA, which is gel-free, quick, and inexpensive. Currently, I focus on the high-throughput gender identification for gentoo penguin (Pygoscelis papua) using the feathers samples. Using Griffiths' P2/P8 primers, molecular sexing failed both in PCR/electrophoresis and in PCR/MCA of gentoo penguin. After the cloning and sequencing, the sex-specific primers for the CHD-Z and CHD-W genes of gentoo penguin, respectively, were redesigned for PCR/MCA. Using this specific primer set, 10 samples of gentoo penguin were examined simultaneously and the Tm peaks of CHD-Z and CHD-W PCR products were able to be distinguished. Therefore, the high-throughput avian molecular sexing technique was successfully applied to gentoo penguin.