桉树EST-PCR产物测序方案的优化

An Optimized Protocol for Sequencing EST-PCR Products in Eucalyptus

EST-PCR产物测序是EST标记开发的重要步骤。本研究利用乙醇-醋酸钠法、清洁试剂盒、虾碱磷酸酶(SAP)-核酸外切酶Ⅰ(ExoⅠ)法和凝胶回收试剂盒4种方法,对6个桉树EST-PCR产物的纯化效果和测序结果进行了比较,推荐乙醇-醋酸钠法为首选的纯化方法;利用测序试剂盒Bigdye TerminatorV3.1,对1/2、1/4、1/8、1/16和1/32标准用量(8.0μL)的测序结果的分析表明,1/16和1/32用量仍可有效测序,均可作为优先的测序试剂用量。同时,也发现EST-PCR扩增谱带单一、明亮的序列与设计的目标EST具有较好的同源性,开发SNP和Indel标记的潜力极大,但较弱的谱带则可能是非特异性扩增的产物。乙醇-醋酸钠法纯化和Bigdye Terminator1/16或1/32用量的测序方案,具有成本低和操作简便的优点,适合96孔板或384孔板大规模EST-PCR产物的测序。

Sequencing of EST-PCR products is important for EST-based marker development. In this study, four procedures for purifying EST-PCR products, namely, ethanol-NaAc, cleanup kit, SAP （shrimp alkaline phosphatase）- Exo Ⅰ （exonuclease Ⅰ）, and gel extraction kit, were compared for their yield and effects on subsequent sequencing, from which ethanol-NaAc was selected as the sound choice. Further, five dosages of Bigdye Terminator V3.1, including 1/2, 1/4, 1/8, 1/16, and 1/32 of the manufacture＇s standard （0.8 μL）, were compared for their sequencing results, from which the last two were recommended in terms of reliability and cost control. In addition, the EST-PCR products, if bright and single in electrophoresis, were highly identical to the original target ESTs and had a great potential in developing SNP and Indel markers, whereas weak EST-PCR products might represent non-specific genomic region to the target EST sequences. The protocol optimized here is advantageous in low cost and technical simplicity and suitable for large-scale sequencing based on 96- or 384- well plates.