Establishment and Optimization of MSAP Reaction System of Astragalus Mongolica
- VernacularTitle:蒙古黄芪MSAP反应体系的建立与优化
- Author:
Fang WANG
1
;
Pengfei ZHANG
;
Hongling TIAN
;
Shuhong GUO
;
Yaling LIU
Author Information
- Keywords: Astragalus mongolica; MSAP; System optimization; DNA methylation
- From: World Science and Technology-Modernization of Traditional Chinese Medicine 2023;25(9):3174-3181
- CountryChina
- Language:Chinese
- Abstract: Objective To study epigenetic diversity of Astragalus mongolicus by methylation-sensitive amplification polymorphism(MSAP)technique,and to establish and optimize the response system of MSAP technique applicable to Astragalus Mongolicus.Methods Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao was used as material.The single factor and orthogonal design method were used to optimize the key influencing factors of the reaction system of Astragalus Mongolian methylation-sensitive amplification polymorphism(MSAP)technique,and the best reaction system of Astragalus Mongolian MSAP was established.Results 0.5 μL each of EcoR I and Msp I/Hpa II were added to the 20 μL double-enzyme digestion reaction system,and the digestion was completed in a water bath at 37℃for 6 h to complete the digestion;the optimal ligation system was 25 μL,including 10 μL digestion product,0.2 μL T4 DNA Ligase,1 μL EcoR I-adapter,1 μL Hpa II/Msp I-adapter,2.5 μL 10×T4 Buffer;25 μL optimal pre-amplification reaction system,including 2.5 μL 10×PCR buffer,0.5 μL dNTP,2.0 μL ligation product,0.3 μL Taq DNA polymerase,1.0 μL upstream and downstream primers;25 μL optimal selective amplification system,including 2 μL 20-fold diluted pre-amplification product,1.0 μL 10×PCR buffer,1.0 μL dNTP,0.2 μL Taq DNA polymerase,0.8 μL primers.Conclusion The optimized MSAP system was detected by polyacrylamide gel electrophoresis,which showed that the polymorphism was good,the system was stable,the bands were clear and repeatable,which laid the foundation for the subsequent MSAP analysis of Astragalus mongolica.
