大花蕙兰基因组DNA提取及RAPD反应条件探索

Genomic DNA Extraction and RAPD Protocols for Cymbidium hybridium

摘要:

用SDS、CTAB和改良CTAB法分别提取大花蕙兰叶片基因组DNA，发现以改良CTAB法提取的DNA纯度高，产率也较高。以5'-GGTGCTCCGT-3'(BA440)为随机引物，并以大花蕙兰品种‘金杯’（Cymbidium hybridium CV．Hiroshima Golden Cup“Sunny Moon”）的DNA为模板，对RAPD（Random Amplified Polymorphic DNA）反应体系进行了优化研究，结果表明,25 μl反心体系中，Mg²⁺、Taq DNA聚合酶、引物、模板DNA和dNTP5种主要成分的适宜浓度或用量分别是:2．0 mmol/L、1.0 U、0．20 μmol/L、25 ng和0.20 mmol/L。扩增程序优化为:94℃预变性4 min；94℃变性0．5 min，37℃退火1 min，72℃延伸1 min，40个循环；最后72℃延伸7 min。

Abstract:

SDS, CTAB and modified CTAB methods were used to extract DNA from the leaves of Cymbidium hybridium cv. Hiroshima Golden Cup “Sunny Moon” and cv. Fortissimo “Pianist”. The results showed that modified CTAB method was best for DNA quality and production. The optimal reaction of RAPD in C. hybridium was studied with primer 5'-GGTGCTCCGT-3' for cv. Hiroshima Golden Cup “Sunny Moon”. The optimum concentration of five important components such as Mg²⁺, Taq DNA polymerase, primer, template DNA, and dNTP in 25 μl RAPD reaction system were 2.0 mmol/L, 1.0 U, 0.20 μmol/L, 25 ng, and 0.20 mmol/L, respectively. Modified thermal profile consisted of an initial denaturation step at 94℃ for 4 min, followed by 40 cycles of 94℃ for 30 s, 37℃ for 1 min and 72℃ for 1 min and a final exposure to 72℃ for 7 min.