荧光标记复合PCR扩增微卫星位点的构建与优化

本研究采用毛细管电泳技术，构建并优化了荧光标记复合PCR同时扩增多个微卫星位点。主要过程为：首先根据设计所扩增微卫星位点的期望长度，将9个微卫星位点分成两组，5个位点用FAM（蓝色）标记，4个位点用HEX（绿色）标记；两种荧光类型分组优化，用琼脂糖胶电泳检测。其次，荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点，采用ABI3730xl毛细管电泳检测，以ROX500（红色）为长度标准物，结果经Genemapper3.5软件 分析，检测结果表明毛细管电泳检测荧光标记复合PCR产物不仅精确读取微卫星位点的长度（分辨率高达1bp），还能区分微卫星位点复制时滑链所引起的“回声斑”；调整各微卫星位点引物比列使所有位点扩增强弱均匀。最后，逐一检测复合PCR基本参数（dNTP浓度、 PCR程序和模版DNA用量）对复合PCR产物的影响，优化PCR。结果表明通过毛细管电泳检测荧光标记复合PCR产物来读取微卫星位点的基因型具有精确性、高效性和稳定性。

Construction and Optimization of Fluorescently Labeled Multiplex-PCR

To amplify microsatellite loci, one fluorescently labeled multiplex-PCR was constructed and optimized using capillary electrophoresis. First, according to the expected length, 9 microsatellite loci were divided into 2 groups , 5 labeled with FAM (Blue) and 4 labeled with HEX (Green). Different fluorescent groups were optimized separately using agar gel electrophoresis. Second, 8 Chinese mitten crabs were was amplified by one set of fluorescently labeled primers（9 loci multiplex-PCR. The Multiplex-PCR products were detected through capillary electrophoresis with ROX500 as size standard and analysised using Genemapper3.5. The results show that capillary electrophoresis has 1 bp precision and can differentiate the main peak and the stutter bands caused by slippage. Uniform PCR products were obtained by adjusting the ratio of primer concentration. Finally, the fundamentals parameters （dNTP concentration、 PCR program and template DNA concentration） were tested one by one. This study showed it was precise, efficient and stable to genotype microsatellite by the detecting the fluorescent multiplex-PCR with capillary electrophoresis.