Cheng Lu, Sun Beili, Sun Yu, Xiao Pengfeng, Ge Qinyu, Zheng Yuanzhuo, Ke Xiaoyan, Zhou Ying, Zhang Minghao, Chen Ping, Lu Zuhong
State Key Laboratory of Bioelectronics, Southeast University, Nanjing, 210096, China.
J Nanosci Nanotechnol. 2010 Jan;10(1):479-86. doi: 10.1166/jnn.2010.1727.
Previously we have developed polyacrylamide gel-based DNA microarray to genotype single nucleotide polymorphism (SNP) in a large number of samples, which has been proved as a simple and robust platform for high-throughput SNP screening. Here we improved this method to detect multiple SNPs by introducing multiplex polymerase chain reaction (multiplex-PCR) and immobilizing the products of multiplex-PCR to fabricate gel-based microarray and applying universal dual-color detectors in hybridization. In this report, five SNPs (rs191296, rs2280073, rs17599165, rs17599416 and rs7660336) of GABRA4 gene were chosen and successfully analyzed with the improved platform. Our experiment demonstrated that 3-dimentional polyacrylamide gel-based microarray of multiplex-PCR products make the platform for multiple SNPs genotyping becoming more labor-saving and time-saving. Appling the universal dual-color fluorescent detectors can reduce the cost over two-thirds for multiple SNPs analysis. It is concluded that the multiplex-PCR combined with the gel-based microarray hybridized with universal dual-color fluorescent detectors is efficient, rapid and simple for the detection of a single nucleotide mismatch, and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays.
此前我们已开发出基于聚丙烯酰胺凝胶的DNA微阵列，用于对大量样本中的单核苷酸多态性（SNP）进行基因分型，该方法已被证明是一个用于高通量SNP筛选的简单且可靠的平台。在此，我们对该方法进行了改进，通过引入多重聚合酶链反应（多重PCR），并将多重PCR产物固定以制备基于凝胶的微阵列，并在杂交中应用通用双色检测器来检测多个SNP。在本报告中，我们选择了GABRA4基因的五个SNP（rs191296、rs2280073、rs17599165、rs17599416和rs7660336），并使用改进后的平台成功进行了分析。我们的实验表明，基于三维聚丙烯酰胺凝胶的多重PCR产物微阵列使多个SNP基因分型平台变得更加省力省时。应用通用双色荧光检测器可将多个SNP分析的成本降低三分之二以上。结论是，多重PCR结合与通用双色荧光检测器杂交的基于凝胶的微阵列，对于检测单核苷酸错配高效、快速且简单，并且在构建DNA微阵列的效率、保真度和成本方面可能具有很强的竞争力。
