多重PCR反应中关键因素和实验步骤

 

琼脂糖凝胶与聚丙烯酰胺凝胶

琼脂糖 . 长度彼此相差 30�40bp 的多重 PCR 产物在通常使用的 SeaKem 或 NuSieve(FMC BioProducts) 3% 的琼脂糖凝胶可以很好的区分开。在低电场强度下过夜电泳可以优化每个 PCR 产物条带的电泳效果，特别是当 PCR 产物小于 400�500 bp 时。

聚丙烯酰胺凝胶 . 为了分离长度仅差几个 bp 的 PCR 产物（例如微卫星标记）需要使用浓度 6%�10% 的聚丙烯酰胺凝胶 (PAA 胶 ) 。非变性 PAA 胶可以有效的区分非多态性基因，但在这种胶上分离微卫星片断会出现不正常条带。例如在分析含有单拷贝人类第 12 号染色体的两个杂交瘤细胞系的人类第 12 号染色体上某个多态性位点时，对于每一个被检测的基因位点，在非变性 PAA 胶上都出现了两个条带 (e.g., Figure 5d) 。在常规的 6% PAA/7 M urea 测序胶上，每个被检测的基因位点都仅有一条带 ( 比较 Figure 5, d 和 e 的产物 ) 。

我们用一系列样品检测了多个参数来优化多重 PCR 。对于任何 PCR 反应，为了获得高特异性的扩增产物，退火温度和 KCl 浓度（盐浓度）的最优组合是最重要的。 Mgcl 2 浓度应当与 dNTP 的量成比例，对于任何反应这个比值可以是常数。虽然逐渐提高 MgCL 2 浓度可以增强反应的特异性，但是看起来退火温度和 KCl 浓度（盐浓度）对于获得高产量的特异性 PCR 扩增产物更为重要。在多重 PCR 中调节每一个基因的引物量也很重要。 Figure 1 给出了进行高效的多重 PCR 的有效方法。尽管无法完全列出影响 PCR 反应的所有因素。然而本研究中所提到的参数的优化提供了解决多重 PCR 中常见问题的基本方法。

参考文献

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• PCR/RT-PCR/qPCR

• 电泳设备
• 紫外设备
• 普通PCR仪
• 定量PCR仪
• PCR/RT-PCR/qPCR试剂
• PCR引物
• PCR试剂
• PCR对照
• 特异性PCR试剂盒
• PCR克隆试剂盒
• RNA
• RNase检测/去除
• RT-PCR试剂
• RT-PCR标准品
• 定量PCR试剂
• 定量PCR标记
• 总RNA分离纯化盒
• PCR产物纯化