亲和素磁珠分离毛竹SSR标记方法的优化

在参考其它文献报道的基础上,构建毛竹SSR位点富集文库。用EcoRⅠ分别和DraⅠ、EcoRⅤ、SmaⅠ、PvuⅡ四种平端限制性内切酶对毛竹基因组DNA进行双酶切,酶切片段回收后与根据抑制性PCR原理设计的接头连接。在利用亲和素磁珠富集含有SSR序列的DNA片段过程中,对杂交、漂洗及洗脱步骤进行优化。通过三引物特异PCR筛选含有SSR序列的阳性克隆并结合测序结果对富集SSR文库进行评价,确定了一条简单、经济、高效分离竹类植物SSR标记的方法。

Optimal isolation of SSR marker in Phylbstachys pubescens by streptavid in-coated magnetic beads

In this experiment,we constructed enriched library that contains SSR loci of Phylbstach- ys pubescens with reference to other reports.In the processs of experiment,genomic DNA isolated from Phylbstachys pubescens was digested with EcoRⅠand other four kinds of blunt ends restriction enzyme DraⅠ,EcoRⅤ,SmaⅠ,PvuⅡseparately.Fragments were purified and ligated with ada- pters which were designed on the basis of suppression-PCR.During the course of enriched DNA fragments which contain SSR by streptavid in-coated magnetic beads,the approach of hybridization, washing and elution was optimized.The library was estimated by percent of positive colon selected by specific PCR which use three kinds of primer and results of sequence.Then we confirmed a method which is simple,economical and efficient to isolate SSR marker from bamboo.