中國(guó)海洋大學(xué)基因工程l4第二章pcr技術(shù)(文件)
【正文】 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 using a suitable Aspecific primer and a bubblespecific primer derived from the strand opposite to that used for making primer B. 3 Figure . AluPCR permits amplification of DNA sequences located between two closely positioned but oppositely orientated Alu repeats. (A) Primers can be designed from the Alu consensus sequence to hybridize to sequences at either terminus of the repeat element, with the
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