三维凝胶NAT2基因分型芯片的制备与优化

Preparation and optimization of gel-based DNA microarray for NAT2 genotyping

目的研制N-乙酰化酶2（NAT2）基因分型芯片，以快速、准确检测患者的NAT2单核苷酸基因多态性。方法设计并合成中国人中常见的NAT2酶基因5个突变位点的探针对和包含5个突变位点的两对引物；样本经PCR扩增后，采用丙烯酰胺点样液制备芯片，并与TEMED反应固化芯片；分别用探针与芯片杂交，采用博奥晶芯LUXSCAN10K微阵列芯片扫描仪分析结果。结果优化了PCR扩增条件，使两组PCR在相同条件下扩增；优化了芯片制备及杂交条件，双链DNA采用碱变性，清除非特异键合采用电泳方法；建立了NAT2酶基因分型标准：信号强度Cy3：Cy5≥5为野生型，Cy3：Cy5为0．6～1．5时为杂合子，Cy3：Cy5≤0．2为突变型；对20例标本的基因分型结果采用直接测序法进行验证，结果均一致。结论三维凝胶NAT2基因点突变分型芯片法是一种特异性强、高通量的NAT2基因分型方法，适用于临床快速基因分型，指导相关药物合理使用。

Aim To establish a 3-D polyacrylamide gel-based microarray method to detect genotypes of Nacetyhransferase2 （NAT2） of patients. Methods The important single nucleotide polymorphisms （SNPs） sequences of NAT2 were searched. Primers and probes were designed and synthesized aided by computer. NAT2 DNA microarray was prepared by dotting the products of PCR, polymerized by TEMED vapor. The products scanned gained by PCR and hybridization were and analyzed. Results NAT2 DNA wicroarray was prepared for the detection of SNPs. The optimization of PCR and hybridization were executed successfully. At the same time, constituted criteria for classification were constructed successfully. The signal intensity ratio of Cy3 to Cy5 for the wild type homozygote is ≥5, and mutant is ≤0. 2, when the ratio is between 0. 6 - 1.5, it is heterozygote. The genotyping results of 20 samples were tested by direct sequencing, genotyping results detected by microarray conformed to the direct sequencing. Conclusion NAT2 DNA microarray demonstrated that this is a reliable novel method for NAT2 genotyping; it can be used in rational drug therapy .