三维聚丙烯酰胺凝胶DNA芯片用于大样本单核苷酸多态分型的方法研究

目的 建立三维聚丙烯酰胺凝胶DNA芯片用于大样本单核苷酸多态(single nucleotide polyrnorphisms,SNP)分型的方法.方法 丙烯酰胺基团修饰的PCR产物与丙烯酰胺单体混合后,在丙烯酰胺基团修饰的玻片上点样进行共聚合,建成三维凝胶DNA阵列;芯片与一对特异探针和一对分别标记了Cy3或Cy5的通用序列标签(Tag1和Tag2)进行杂交;杂交后用施加电场的方法去除非特异吸附和错配,最后通过双色荧光共聚焦扫描进行SNP分型.结果 3-D凝胶芯片不但具有很高的固定效率,而且可以提供高效的杂交环境;通用序列标签的使用木需对每个位点都标记荧光,使检测成本大幅降低;外加电场使得单碱基错配容易识别,且能显著降低芯片的信噪比.结论 基于3-D凝胶的基因芯片技术用于大样本SNPs分型简单易操作,且高通量、高特异性、低成本,该方法将可以更广泛地应用于不同需求的DNA检测中.

Study on the genotyping of single nucleotide polymorphisms for a large number of samples by three-dimensional polyacrylamide gel-bused microarray method

Objective To genotype single nucleotide polymorphisms (SNPs) in a large number of samples by applying three-dimensional polyacrylamide gel-based microarray. Methods The method relies on copolymerization of acrylamide-modified PCR products with acrylamide monomers and acryl-modified slides to prepare gel-based microarray. Then array is hybridized with a pair of specific probes and the two universal dual-color fluorescent detectors labeled with Cy3 or Cy5 respectively (Tag1 and Tag2). Electrophoresis is used in post-hybridization to remove the nonspeeifically bound targets and mismatches. Finally, genotyping is based on the images captured through two-color fluorescent scanning. Results The 3-D gel-immobilization of nucleic acids has a high immobilization yield and good hybridization efficiency. As universal dual-color fluorescent detectors are used, it is not required that specific probes be labeled for all SNPs, therefore the expense for synthesis can be reduced considerably. Electrophoresis in post-hybridization can enhance the capability for discriminating a single nucleotide mismatch from the perfectly matched sequence and improve the signal-to-noise ratio significantly. Conclusion The gel-based microarray is a rapid, simple and high-throughput method for SNPs genotyping and may be very competitive in the efficiency, fidelity and cost for constructing DNA microarrays, which will hold significant promise for applications in human DNA diagnostics.