鹿茸干细胞MSAP分析技术体系的建立及引物筛选

Establishment of MSAP Analysis System and Selection of Primers for Antler Stem Cell

利用甲基化敏感扩增多态性(Methylation sensitive amplified polymorphism,MSAP)技术进行鹿茸干细胞基因组DNA甲基化研究,建立并优化鹿茸干细胞MSAP技术分析的反应体系。对MSAP技术中的关键步骤DNA提取、酶切、预扩增反应中连接产物稀释倍数、选择性扩增反应中预扩增产物稀释倍数等条件进行了优化。同时,利用优化条件,应用ABI3730xl测序仪对64对引物进行筛选。结果表明:优化后的反应体系得到条带清晰、重复好和特异性强的选择扩增产物。建立的MSAP反应体系保证了MSAP图谱的稳定性和多态性,筛选出来的32对引物组合满足鹿茸干细胞以及后续鹿相关基因组DNA甲基化研究。

In this study,we investigated genome-wide DNA methylation in antler stem cells,and established methylation sensitive amplified polymorphism（ MSAP） system. The key factors influencing the results of MSAP system were comparetively analyzed. DNA extraction,reaction time of restricted enzymes,diluted multiples of adapter-ligation DNA for pre-amplification products,and diluted factor of pre-amplification products for selective amplification were optimized. Meanwhile,the established MSAP conditions were used to conduct a preliminary polymorphism screeing on 64 pairs of primers.The results showed the clear bands,specificity and polymorphism of selective amplification product could be obtained in the established MSAP system. Clear and stable MSAP patterns were achieved,and 32 pairs of primers were selected for MSAP analysis. These results provide fundamental reference for further analysis of genome-wide DNA methylation on antler stem cells and deer.