中国海洋大学学报（自然科学版）2017，Vol.47Issue(11)：47-52,6.DOI:10.16441/j.cnki.hdxb.20160302
电洗脱法和透析在紫菜腐霉PacBio全基因组测序DNA样品纯化中的应用
The Applications of Electroelution and Dialysis to Purify Genome DNA of Pythiumporphyrae
摘要
Abstract
PythiumPorphyraeis a special pathogen to infect Nori,which caused red rot disease in Pyropiayezoensisfrequently.It brings seriously reduce in Nori production and quality every year in China,Korea and Japan.So,more genetic information about the pathogen could help us exploit disease prevention and control measures.The most useful and quickly method to get the genetic information is carrying on its whole genome analysis.High quality DNA sample is the key work for carrying on the project.Particular habitation and metabolism and abundant secondary metabolites in cells of marine organism often affects the quality of the genome DNA.The P.porphyraegenome DNAwas polluted by RNA seriously in our preliminary experiment.Usually,RNA was easily degradation by RNase A,but the RNA which residual in P.porphyrae genome DNA was not removed absolutely,even by extending RNase A degradation time and increasingRNase A degradation times,RNA pollution in the DNA simple cannot be completely degradation,the DNAlost and degradation seriously,butthe DNA simple qualitystill cannot reach up to the standard for constructing PacBio libraries.The librarywhich was constructed mandatory cannot sequence normally.In this study,DNA was obtained by EDTA methods,then the agarosegel electrophoresis (AGE) was used to separate genome DNA and RNA.The DNA bandswere cut under UV light quickly andwere transferredinto dialysis bag,further electrophoresisto insure the direction of the DNA band migrating as before.Stopping electrophoresis after the DNA band disappeared from the agarose gelby checking under UV light.This result showed that the DNA in agarose gel had enteringto the TAE solution of the dialysis bag absolutely.The dialysis which contained DNA and agarose gel,was put into 1 liter of TE buffer,the sodium acetate (CH3COONa) in the dialysis would moved from the dialysis bag to the TE buffer.At last,the pure TE solution of DNAwas got eventually.Enriching DNA by anhydrous ethanol and then detecting the DNA by AGE and pulsed field gel electrophoresis (PFGE).The results showed that the DNA band was clearly,no RNA pollution and the DNA was integrity.It can be used for PacBio libraries constructing and sequencing.关键词
紫菜腐霉/基因组DNA/RNA污染/透析Key words
Pythiumporphyrae/genome DNA/RNA pollution/electroelution/dialysis分类
生物科学引用本文复制引用
邹丹丹,唐祥海,莫照兰,茅云翔..电洗脱法和透析在紫菜腐霉PacBio全基因组测序DNA样品纯化中的应用[J].中国海洋大学学报（自然科学版）,2017,47(11):47-52,6.基金项目
国家自然科学基金项目(31372517) （31372517）
国家高技术研究计划项目(2012AA10A406) （2012AA10A406）
山东省自主创新专项(2013CXC80202)资助 Supported by National Natural Science Foundation of China (31372517) （2013CXC80202）
National High Technology Research Projects (2012AA10A406) （2012AA10A406）
Independent Innovation Foundation of Shandong Province (2013CXC80202) （2013CXC80202）
