摘要
探讨DNA提取中不同蛋白质变性剂对胞嘧啶(C)和5-甲基胞嘧啶(5mC)色谱的影响,建立蝴蝶兰类原球茎DNA总甲基化水平的HPLC测定方法。本实验采用2种CTAB法(蛋白质变性剂不含酚和含酚)提取基因组DNA,水解后分别以反相HPLC测定C和5mC含量。色谱条件为:色谱柱HypersilBDS Cl8,甲醇-10mmol/L戊烷磺酸钠-三乙胺(6∶90∶0.2,V/V)为流动相,流速0.5ml/min,检测波长273nm。应用不含酚变性剂所提取DNA的水解液C和5mC能较有效分离。以本试验优化的DNA提取方法和HPLC色谱条件,基因组DNA水解液的C和5mC色谱可得到较好的效果。
The experiment aimed to explore the effect of different protein denaturant applied in DNA extraction on the chromatogram of cytosine（C） and methylcytosine（5mC）,and establish a HPLC method for determining the total level of DNA methylation in PLB of Phalaenopsis spp.Genomic DNA was extracted by two kinds of CTAB method（the protein denaturant was tested with phenol or without phenol）.The contents of C and 5mC in two kinds of DNA hydrolyzate were determined with RP-HPLC,respectively.HPLC conditions were Hypersil C18 BDS column,mobile phase consisting of methanol-pentane sodium（10mmol/L）-triethylamine（10-90-0.2,V/V） at a flow rate of 0.5ml/min,and the detection wavelength 273nm.The separation results of both C and 5mC in the DNA extracted through the protein denaturant without phenol（CI） were more effective.With the opposite method of DNA extraction and HPLC conditions,the better result was achieved in the chromatogram of C and 5mC in genomic DNA hydrolyzate.
出处
《核农学报》
CAS
CSCD
北大核心
2010年第4期752-756,共5页
Journal of Nuclear Agricultural Sciences
基金
福建省自然科学基金资助项目(C0410040,2009J01079)
