应用不对称PCR技术提高寡核苷酸基因芯片杂交效率

Enhancing the hybridization efficiency of oligo microarray by asymmetric PCR

目的:比较采用常规PCR和不对称PCR分别进行样品的掺入荧光标记,应用于60mer寡核苷酸芯片杂交,分析其对芯片杂交效率的影响。方法:以A/T克隆分别将NS1、NS2a、NS2b、NS4a和NS4b基因片段与pMD18-T载体连接,应用常规PCR和不对称PCR两种方法进行掺入荧光标记,将荧光标记样品与寡核苷酸芯片杂交,在相同条件下完成杂交清洗和芯片的扫描检测。结果:与常规PCR标记方法相比,采用不对称PCR技术标记单链样品与寡核苷酸芯片杂交,能提高芯片的杂交效率。结论:应用不对称PCR技术对样品进行荧光标记后,与寡核苷酸芯片杂交能提高芯片的杂交效率,有利于检测型寡核苷酸基因芯片的应用。

Objective：To study the hybridization efficiency of two fluorescence labeling methods by oligo microarray hybridization. nethod：Gene fragments of NS1,NS2a, NS2,NS4a and NS4b were ligated with pMD18-T vector by A/T clone. Then they were labeled by traditional PCR and asymmetric PCR. The labeled samples were examined by the oligo microarray, followed by washing and scanning under the same conditions. Results： Comparing to the traditional PCR labeling method, the asymmetric PCR labeling method showed superior results with enhanced hybridization efficiency. Conclusion：The hybridization efficiency can be enhanced by asymmetric PCR labeling method, which improving the applicability of oligo microarray technology.