野生白及SRAP-PCR反应体系的优化  

Optimization of SRAP-PCR Reaction System of Bletilla striata

作　　者：蒋瑞彬[1] 徐旭栋[1] 蓝小明[1] 王鸯妮[1] 丁志山[1] 金波[1] 

机构地区：[1]浙江中医药大学,浙江杭州310053

出　　处：《中华中医药学刊》2013年第2期353-355,I0015,共4页Chinese Archives of Traditional Chinese Medicine

基　　金：浙江省教育厅科研基金资助项目(71003012);浙江省中医药青年基金资助项目(A2005Y005);浙江中医药大学学生科研基金资助项目(2012)

摘　　要：采用浓度梯度设计法研究适合白及的SRAP-PCR反应体系。对影响SRAP-PCR的DNA模板、引物、dNTP、Mg2+、Taq酶、退火温度等6个因素进行优化。最终确定白及的SRAP-PCR优化体系:在20μL体系中,DNA模板30 ng、Mg2+2.5 mmol/L、dNTPs 0.25μmol/L、引物20μmol/L、Taq酶2.0U,扩增体系为:94℃预变性5 min;5个循环的94℃变性1 min,36℃退火1 min,72℃延伸1 min;之后35个循环:94℃变性1 min,48℃退火1 min,72℃延伸1 min;72℃最后延伸5 min。该体系的建立为野生白及种质资源遗传多样性的进一步相关遗传分析奠定了基础。The concentration gradient was used to optimize reaction system for sequence - related amplified polymorphism（SRAP） of Bletilla striata. The suitable concentration of template DNA, primer, Mg^2＋ , dNTPs, rTaq DNA polymerase and annealing temperature were investigated through single factor test. The SRAP - PCR amplifying system was optimized which came out total 20 μL reaction system containing： 30ng DNA template,2.5 mmol/L Mg^2＋ ,0.25 μmol/L dNTPs ,20 μmol/L primers ,2.0U Taq DNA polymerase, the most suitable PCR amplification procedure was pre - denaturing at 94 ℃ for 5 min;5 cycles of denaturing at 94 ℃ for 1 min, annealing at 36 ℃ for 1 min and extending at 72 ℃ for 1 min, then 35 cycles of denaturing at 94 ℃ for l min, annealing at 48℃ for 1 min and extending at 72℃ for 1 min, extending at 72 ℃for 10 min finally. The optimized system laid the groundwork for SRAP molecular analysis and further research on BletiUa striata.

关 键 词：白及 SRAP—PCR 体系优化 

分 类 号：R284.3[医药卫生—中药学]