Tan Guihong, Gao Yin, Shi Miao, Zhang Xinyue, He Shanping, Chen Zhangliang, An Chengcai
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University Beijing 100871, China.
Nucleic Acids Res. 2005 Aug 2;33(13):e122. doi: 10.1093/nar/gni124.
In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primer and a vector primer. However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could not be screened out. This simple method proved to be efficient, reliable, inexpensive and time-saving, and may be suitable for the molecules for which gene-specific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosome walking and obtained 16 positive results from 17 samples.
在本文中，我们提出了一种用于基因或染色体步移的新型PCR方法，称为位点寻找PCR。该PCR由位点寻找器在低温下引发，然后用基因特异性引物和位点寻找器引物对目标分子进行指数扩增，并用另一个基因特异性引物和载体引物进行筛选。然而，由于茎环结构的抑制作用，非目标分子无法进行指数扩增，也无法被筛选出来。这种简单的方法被证明是高效、可靠、廉价且省时的，可能适用于有基因特异性引物的分子。更重要的是，使用这种方法可以轻松获得大片段DNA。为了证明位点寻找PCR的可行性和效率，我们采用该方法进行染色体步移，从17个样本中获得了16个阳性结果。
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