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[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。
[Objective] The research aimed to study the extraction of genomic DNA and the establishment and optimization of its RAPD system. [Method] The genomic DNA was extracted by conventional phenol / chloroform extraction method and the optimum factors affecting the RAPD reaction were optimized. The optimal RAPD reaction system and program were established. [Result] The DNA obtained by conventional phenol / chloroform extraction could fully meet the requirements of RAPD analysis. A set of stable RAPD reaction system suitable for calculi was established by optimizing the reaction system. The total volume of reaction system was 25μl, including 10 × buff-er 2.5μl, 22mmol / L MgCl 2, 0.15mmol / L dNTPs, 0.2μmol / L primer, ng, Taq enzyme 1 U. The amplification procedure was as follows: pre-denaturation at 94 ° C for 5 min followed by 45 PCR cycles (denaturation at 94 ° C for 45 s, annealing at 36 ° C for 45 s, extension at 72 ° C for 2 min and extension at 72 ° C for 10 min. 40 primers for amplification and found that some of the primers could produce stable and clear bands. [Conclusion] This system laid the foundation for the genetic diversity and related molecular markers of Lithocarpus.