To improve the specificity of PCR in detection of genes in genomic DNA, a modified touchdown PCR method was designed by using the common Taq DNA polymerase and the Pfu DNA polymerase with proofreading ability. Genomic DNA was extracted from Dunaliella bardawil, a unicellular and wall-less alga being able to propagate in solution of high concentration of NaCl. Two kinds of PCR using Taq and Pfu DNA polymerases were carried out, utilizing the genomic DNA mentioned above as a template, to amplify the upstream promoter sequence of the cbr (carotene biosynthesis-related) gene. Traditional PCR was used as control. Electrophoresis of the PCRs using Taq DNA polymerase demonstrated that TD-PCR produced a single band of 1 272 bp which was corresponding to the expected cbr promoter, in contrast, however, in the lane of common PCR, two extra nonspecific bands appeared as well as the expected 1 272 bp band. PCRs using the Pfu DNA polymerase displayed that common PCR program generated two bands of 1 272 bp and 500 bp, while the TD-PCR program brought about only the expected 1 272 bp band. PCRs using modified touchdown PCR program with both kinds of polymerase were of higher specificity than common PCR. The modified TD-PCR program can efficiently improve the specificity of PCR. Similar programs are expected to clone gene fragments that are difficult to be cloned by the common PCR method, or to raise the specificity of PCR in which the false positives are difficult to be eliminated.%为提高基因组DNA中的基因PCR检出的特异性,设计了一种改良的降落PCR程序,并分别用TaqDNA聚合酶及高保真Pfu DNA聚合酶进行实验.自盐藻Dunaliella bardawil中提取基因组DNA作为PCR模板,使用Taq DNA聚合酶及Pfu DNA聚合酶,运用普通PCR和降落PCR程序,扩增胡萝卜素生物合成相关基因(cbr)上游启动子序列,并电泳比较PCR扩增产物的特异性.结果显示,使用普通Taq酶PCR,普通PCR程序产生200bp,500bp和1272 bp长的三条带,而TD-PCR程序仅克隆出1272bp的特异带;利用高保真的Pfu DNA聚合酶作PCR,在TD-PCR泳道中仅有1272bp一条带,而普通PCR除了1272bp的特异带外,还出现一条500bp的非特异带.无论使用普通Taq酶或高保真酶Pfu,改良的降落PCR程序均明显提高PCR的特异性,类似的降落PCR程序可望用于克隆用普通PCR难以克隆的基因片段,或在假阳性难以去除的情况下提高PCR的特异性.
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