Wang Hong-min, Ma Wen-li, Huang Hai, Xiao Wei-wei, Wang Yan, Zheng Wen-ling
Institute of Molecular Biology, First Uniform Medical University, Guangzhou 510515, P.R. China.
J Biochem Mol Biol. 2004 May 31;37(3):356-61. doi: 10.5483/bmbrep.2004.37.3.356.
To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.
为开发一种能大规模快速制备DNA微阵列探针的简化方法，构建了一个用于微阵列探针收集的λ噬菌体基因组DNA片段文库。测试并比较了从第一轮PCR产物中回收DNA条带的四种方法。通过一种由凝胶分离第一轮PCR产物介导的新型巢式PCR方法收集DNA微阵列探针。该方法被命名为GIN-PCR。用这种GIN-PCR技术制备的探针作为样本制作DNA微阵列。结果表明，木质牙签优于其他三种方法，因为该技术在第一轮PCR后能稳定地转移DNA条带作为第二轮PCR的模板。成功收集了一组探针，并使用这些探针构建了DNA微阵列。杂交结果表明，这种DNA回收和探针制备技术快速、高效且有效。我们开发了一种经济高效且劳动强度较低的方法，通过电泳后用木质牙签转移凝胶中的DNA条带介导的巢式PCR来制备DNA微阵列探针。
