碱煮法:一种制备海洋浮游生物PCR模板DNA的实用方法

Alkaline-heating method:an effective approach for preparing PCR template DNA from marine plankton

介绍了一种制备海洋浮游生物PCR模板DNA的方法———碱煮法。取少量海水样品(40μL) ,直接经0.25mol/LNaOH和99℃温育处理裂解细胞 ,从而得到环境样品总DNA。实验证明 ,该方法制备的模板DNA可用于细菌核糖体RNA、浮游植物叶绿体rbcL和浮游动物线粒体COI等基因的PCR扩增。但是 ,在使用通用引物扩增细菌基因时 ,假阳性很难避免。该法制备的模板DNA适用于扩增非细菌基因或者特异引物界定的细菌基因。该方法较传统制备方法快速、简便 ,样品需要量减少 。

In this study alkaline-heating,an efficient method for preparing PCR template DNA from marine plankˉton,was developed.A small volume of surface seawater(40μL)was sampled and total DNA was extracted directly by lysing cells using0.25mol/L NaOH and incubating at99℃.The experiment shows that the DNA prepared can serve as PCR template directly for the amplification of genes like ribosomal RNA gene of bacterioplankton,chloroplast rbcL gene of phytoplankton and mitochondrial cytochrome oxidase gene of zooplankton.However,false-positives can not be avoided when bacterial ribosomal RNA gene was amplified with universal primers.Therefore,template DNA prepared using alkaˉline-heating is practicable to amplify genes of non-bacterial or bacterial gene confined by specific primers.Alkaline-heating is a simple and rapid method where only very small sample volume was needed compared with traditional DNA exˉtracting method.It is suitable for the study of planktonic molecular diversities.