中國海洋大學(xué)基因工程l4第二章pcr技術(shù)(存儲版)
【正文】 ? 引物比例 1： 100 ? 表達(dá)子 PCR(expressioncassette PCR ECPCR） ? 酶切位點 克隆 ? 噬菌體啟動子 單鏈探針 ? 蛋白質(zhì)結(jié)合位點 產(chǎn)物純化、檢測 PCR技術(shù) 點突變 APCR ? 反向 PCR(inverse PCR） ? 原位 PCR ? RAPD(Random Amplification Polymorphic DNAs) ? 原理 引物 條件 應(yīng)用 ? 其它 ? 七、 PCR擴增產(chǎn)物的分析 ? 凝膠電泳 ? 雜交 ? 微孔板夾心雜交法（特異性高） :通過一固定于微孔板的捕獲探針與 PCR產(chǎn)物的某一區(qū)域特 異雜交使產(chǎn)物間接地固定于微孔板上，然后，再 利 用一生物素 等非放射性標(biāo)記物標(biāo)記的檢測探針與產(chǎn)物的另一 區(qū)域的 特異性 進(jìn)行 一次 雜交 ， 顯色 后 即可判斷結(jié)果 . 反向 PCR Figure . Vectorette (bubble linker) PCR permits amplification of uncharacterized sequences flanking a known DNA sequence. Note that the vectorettes contain plementary sequences at their ends but unrelated sequences within the bubble. A restriction fragment containing a known sequence A flanked by uncharacterized regions X and Y is ligated to a vectorette linker containing a suitable overhang at one end. PCR amplification of the uncharacterized sequence X is then possible using a primer specific for the known DNA sequence (A) and a primer specific for one of the strands of the bubble in the vectorette linker (B). The vectorette linker primer B cannot prime DNA synthesis initially as there is no sequence to which it can bind: it is identical, not plementary in sequence to one strand of the bubble, and unrelated in sequence to the other. However, primer A initiates synthesis of a plementary DNA strand which will contain a sequence plementary to one of the unique sequences within the bubble linker. As a result, primer B can bind to this newly synthesized strand and initiate new strand synthesis to start a PCR reaction. The flanking sequence Y can similarly be isolated in another reaction
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