SCoT-PCR Primer Selection and Reaction System Optimization of DNA of Marchantia convoluta in Guangxi
10.6039/j.issn.1001-0408.2018.10.04
- VernacularTitle:广西产拳卷地钱DNA的SCoT-PCR引物筛选及反应体系优化
- Author:
Fengfeng XIE
1
;
Peng LI
;
Li LI
;
Hua ZHU
;
Xiaohong SHANG
;
Pingfeng LI
Author Information
1. 广西中医药大学科学实验中心
- Keywords:
Marchantia convolute;
SCoT;
PCR;
Primer selection;
System optimization
- From:
China Pharmacy
2018;29(10):1309-1312
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To establish PCR reaction system of DNA of Marchantia convoluta in Guangxi marked with SCoT polymorphism marker technique by screening primer and optimizing reaction condition. METHODS:Modified CTAB method was used to extract DNA of M. convolute from Guangxi;gel electrophoresis and UV spectrophotometry were used to investigate purity and concentration of DNA. Using sample DNA as template,PCR amplification of 36 SCoT primers was conducted,and suitable primers were screened after electrophoresis,staining and imaging of products. The orthogonal experiment of 5 factors and 4 levels was conducted by 5 main factors as DNA concentration,Mg2 + concentration,dNTP concentration,primer concentration,Taq DNA polymerase concentration. The condition of SCoT-PCR reaction system was optimized. RESULTS:Extracted sample DNA bands were neat without RNA contamination,degradation or dispersion of fluorescence;sample well was clear. UV absorbance ratio ranged 1.7-2.0 at 260 nm and 280 nm;purity and concentration of DNA were both suitable for follow-up test. PCR results of 36 primers showed that product band of No. 4 primer was neat without diffuse fluorescence but with best luminance,so No. 4 primer was used for PCR reaction. The optimal SCoT-PCR reaction system contained 30.00 μg/mL DNA,2.00 mmol/L Mg2+,0.20 mmol/L dNTP, 0.40 μmol/L primer,0.50 U/mL Taq DNA polymerase(total reaction volume of 20 μL). CONCLUSIONS:Suitable SCoT-PCR primer of DNA is screened,and reaction system is optimized. It provides technologic basis for variety identification and genetic relationship analysis of M. convoluta in Guangxi.
