摘要
采用正交设计法,从dNTPs浓度、引物浓度、Mg2+浓度、Taq DNA聚合酶用量4个因素3个水平出发,优化设计圆尾鲎DNA的PCR反应体系(引物为中国鲎微卫星引物).并采用直观分析方法分析正交试验结果,最终建立了圆尾鲎SSR-PCR最佳反应体系:总体积20μL,Taq DNA聚合酶1.5 U、dNTPs 0.16 mmol/L、引物0.2μmol/L、Mg2+2.0 mmol/L;并通过PCR梯度实验进一步优化模板DNA质量浓度、退火温度及退火时间,获得最佳反应条件:模板DNA质量浓度为30 ng/μL,退火温度为48℃,退火时间为20～25 s.对最佳反应体系和反应条件进行了检验,结果显示该反应体系稳定性高、重复性好.
SSR-PCR reaction system in Carcinoscorpius rotundicauda using microsatellite primers from Tachypleus tridentatus was constructed and optimized by employing L9（34）orthogonal design with three levels and four factors,which were the concentrations of dNTPs,primers,Mg2＋ and TaqDNA polymerase.A pair of microsatellite primers from Tachypleus tridentatus was used to amplify microsatellites in Carcinoscorpius rotundicauda.Finally,the optimal SSR-PCR reaction system in Carcinoscorpius rotundicauda was established in 20μL reaction volume,containing 1.5 U TaqDNA polymerases,0.16 mmol/L dNTPs,0.2 μmol/L primers and 2.0 mmol/L Mg2＋.Furthermore,the optimal reaction condition with the optimal concentration（30 ng/μL） of template DNA,optimal annealing temperature（48 ℃）and annealing time（20~25 s）was obtained by gradient PCR reaction.Verification test of the optimized SSR-PCR reaction system and amplificantion procedures showed that this amplification system was stable and practicable.
出处
《集美大学学报（自然科学版）》
CAS
2012年第2期89-95,共7页
Journal of Jimei University：Natural Science
基金
福建科技厅F5类项目(2008F5038)
浙江海洋学院"海洋渔业科学与技术"省重中之重学科开放课题(20100210)
关键词
圆尾鲎
中国鲎
微卫星引物
PCR
正交优化
Carcinoscorpius rotundicauda
Tachypleus tridentatus
microsatellite primers
PCR
orthogonal optimization
