几种海洋微藻基因组DNA的分离提取及PCR检测

Genomic DNA extraction from several marine microalgae species and their examination by polymerase chain reaction(PCR)

用改良CTAB法分别提取6种海洋微藻的基因组DNA,发现提取的DNA纯度、产率高(>100μg.g-1鲜重),DNA完整性好。以6种海洋微藻的基因组DNA为模板,并以5'TTCGAGCCAG3'(OPC-01)为随机引物,对PCR反应条件进行研究。结果表明,25μl反应体系中,Mg2+、Taq DNA聚合酶、引物、模板DNA和dNTP 5种主要成分的适宜浓度或用量分别是:2.0mmol.L-1、1.6U、20pmol.L-1、50ng和2.5mmol.L-1。扩增程序优化为:94℃预变性5min,94℃变性1min,36℃退火1min,72℃延伸2min,循环45次,最后于72℃再延伸10min。酶切实验结果表明,6种海洋微藻的基因组DNA都能被EcoRⅠ酶切,酶切图谱呈弥散状,满足分子水平操作的要求,可直接应用于进一步的分子生物学实验。

A modified CTAB （hexadecyltrimethylammonium bromide） method was used to extract genomic DNA from six marine microalgae species, Heterogloea sp. , Platymonas peculate, Chaetoceros calcitrons, Isochrysis gabana, Chlorella pyrenoidosa and Pavlova. The results showed that the modified CTAB method was very good for extraction of high quality and production of DNA. The optimal reactive conditions of PCR in the six DNA samples were studied with primer 5＇TTCGAGCCAG3＇. The optimum concentrations of five important components, including Mg^2＋ , Taq DNA polymerase, primer, template DNA, and dNTP were 2.0mmol · L^-1, 1.6U, 20pmol · L^-1, 50ng, and 2.5mmol · L^-1, respectively in 25μL PCR reaction system. The modified thermal profile consisted of an initial denaturation step at 94℃ for 5 min, followed by 45 cycles of 94℃ for 1min, 36℃ for 1 rain and 72℃ for 2 rains and a final exposure to 72℃ for 2 min. The enzyme digestion results showed that the six DNA samples could be digested by EcoR I , and were suitable for further molecular biological experiments.