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
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摘要: 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。Abstract: Objective: To study the hybridization efficiency of two fluorescence labeling methods by oligo microarray hybridization. Method: 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.
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