石鲽基因组DNA的提取及其RAPD体系的优化

目的]研究石鲽基因组DNA的提取及其RAPD体系的建立和优化。方法]以石鲽为试材,按常规酚/氯仿抽提法提取其基因组的DNA,对影响RAPD反应的各因素进行优化,建立了石鲽的最佳RAPD反应体系和程序。结果]采用常规酚/氯仿抽提法获得的DNA完全能够满足RAPD分析的要求。通过优化建立一套适合石鲽的稳定的RAPD反应体系:反应体系总体积为25μl,包括10×buff-er2.5μl,MgCl22 mmol/L,dNTPs 0.15 mmol/L,引物0.2μmol/L,模板30 ng,Taq酶1 U。扩增程序为:94℃预变性5 min,45次PCR循环(94℃变性45 s,36℃退火45 s,72℃延伸2 min和72℃延伸10 min。利用该体系对OPK和OPV系列共40条引物进行扩增,发现其中部分引物能产生稳定、清晰的条带。结论]该体系为石鲽遗传多样性以及相关分子标记的研究奠定了基础。

DNA Extraction from Kareius bicoloratus Genome and Its Optimization of RAPD Reaction System

Objective] The study aimed to DNA extraction from Kareius bicoloratus genome and the establishment and optimization of RAPD reaction system.Method] The genomic DNA was extracted from K.bicoloratus individual according to conventional extraction method of phenol/chloroform.Based on the optimization of RAPD reaction conditions,the RAPD reaction system for K.bicoloratus was established.Result] DNA obtained by using the conventional extraction method of phenol/chloroform could fully meet the demand of RAPD analysis.A RAPD reaction system for K.bicoloratus was established through optimization,which was following: the total volume of RAPD reaction was 25 μl,including 2.5 μl 10×buffer,2 mmol/L MgCl2,0.15 mmol/L dNTPs,0.2 μmol/L primer,30 ng template DNA and 1 U Taq DNA polymerase.The PCR program was as follows: 5 min pre-amplification denaturation at 94 ℃,45 cycles of 45 s at 94 ℃ for denaturation,45 s at 36 ℃ for annealing,2 min at 72 ℃ for extension,finally extension at 72 ℃ for 10min.When this system was used to amplify 40 primers from OPK and OPV series,it was found that their part primers could produce the stable and clear bands.Conclusion] This system laid the foundation for further study of the genetic diversity and related molecular markers in K.bicoloratus.