Chong K Y, Chen C M, Choo K B
Department of Medical Research, Veterans General Hospital, Taipei, Taiwan, Republic of China.
Biotechniques. 1993 Apr;14(4):575-8.
We describe here a simple and rapid method for enzymatic DNA amplification using DNA template recovered from membrane filters previously used in hybridization analysis. This is done by first solubilizing membrane pieces carrying DNA of interest in dimethyl sulfoxide, followed by isopropanol precipitation and polymerase chain reaction amplification. The source of membrane-bound DNA successfully tested includes plasmid and human leukocyte DNA and DNA immobilized on bacterial colony filters and plaque lifts. The sensitivity of the procedure is such that DNA recovered from 0.5 microgram of filter-bound total human DNA was enough for PCR amplification of a 0.3-kb fragment. Our protocol will be useful for recycling of scarce DNA samples for cloning and sequencing purposes.
我们在此描述一种简单快速的酶促DNA扩增方法，该方法使用从先前用于杂交分析的膜滤器中回收的DNA模板。具体做法是，首先将携带感兴趣DNA的膜片溶解于二甲基亚砜中，随后进行异丙醇沉淀和聚合酶链反应扩增。成功测试的膜结合DNA来源包括质粒、人白细胞DNA以及固定在细菌菌落滤膜和噬菌斑影印膜上的DNA。该方法的灵敏度很高，从0.5微克滤膜结合的总人DNA中回收的DNA足以用于0.3千碱基片段的PCR扩增。我们的方案对于回收稀缺的DNA样本用于克隆和测序目的将很有用。
