利用同步聚合酶連鎖反應進行乳牛之性別鑑定

本研究旨在利用同步聚合酶連鎖反應（real-time polymerase chain reaction, real-time PCR），配合SyberGreen（SYBR）與螢光標定的探針（probe）進行乳牛的性別鑑定，以探討real-time PCR應用之可行性。利用純化之不同性別乳牛基因組DNA與體外培養（in vitro culture）之荷蘭母牛卵丘細胞或公牛耳朵成纖維細胞做為性別鑑定之樣品，先經蛋白酶K（proteinase K）前處理後利用ZFX/ZFY（X/Y-linked zinc finger protein）引子進行傳統PCR預先擴增（pre-amplification），獲得PCR產物後設計nested PCR引子並應用SYBR進行real-time PCR。結果顯示隨著起始模板DNA濃度之增加，PCR出現擴增曲線的循環較早。雖然如此，但在不同PCR循環所獲得的產物均具有專一性且重疊的DNA解離曲線（dissociation curve），顯示PCR擴增產物之高度特異性。預先擴增初次PCR產物配合nested PCR引子及性別特異性的TaqMan螢光探針進行雜合反應（hybridization），由反應產生的不同螢光將不同性別樣品分群而判定其性別。試驗結果顯示，待測之基因組DNA或1到20個不同數量的體細胞樣品在雜合反應後因釋放螢光之不同而分群，有效完成性別鑑定。本研究顯示，應用real-time PCR進行乳牛的性別鑑定，可直接判定樣品之性別而不需進行傳統之電泳分析，且有較高的敏感性與重覆性，為具應用潛力的乳牛性別鑑定方法。

關鍵字

性別鑑定；乳牛；探針；同步聚合酶連鎖反應

並列摘要

The purpose of this study was to develop a fast real-time polymerase chain reaction (PCR) method for sexing dairy cattle. DNA samples prepared from peripheral blood and in vitro cultured somatic cells were pre-amplified by traditional PCR with ZFX/ZFY primers. Resulted products were used as a template in the followed real-time PCR assay, with nested primers and SYBR, and then determined with sex specific fluorogenic probes for ZFX and ZFY. The results showed that the higher the concentration of initiation template was, the earlier the amplification plots appeared. The dissociation curve of real-time PCR products showed high specificity. By using SYBR analysis and sex specific fluorogenic probes, the accuracy, efficiency, and reproducibility of sex determination for bovine genomic DNA and somatic cells were significantly improved. In addition, the time-consuming electrophoresis was eliminated by using fluorogenic probes for sexing dairy cattle.

並列關鍵字

Sexing ； Dairy cattle ； Probe ； Real-time PCR ； ZFX (X-linked zinc finger protein)

主題瀏覽