[Objectives]To establish a non-toxic and efficient method for extracting DNA and total RNA from peanuts and laying a solid foundation for the molecular biology study of peanuts.[Methods]Based on the principle and meth...[Objectives]To establish a non-toxic and efficient method for extracting DNA and total RNA from peanuts and laying a solid foundation for the molecular biology study of peanuts.[Methods]Based on the principle and method of purifying nucleic acids by silica gel adsorption at high salt and low pH condition,a non-toxic and efficient method to extract peanut DNA and total RNA using cetyltrimethyl ammonium bromide(CTAB)extraction solution was designed.The quality and purity of nucleic acids were detected by agarose gel electrophoresis and nucleic acids protein analyzer,respectively.The quality of DNA was further verified by enzyme digestion and PCR amplification using molecular marker techniques.The quality of total RNA was further verified by reverse transcription(RT)-PCR of actin gene and cDNA-SCoT gene differential display technique.[Results]The agarose gel electrophoresis test showed that the peanut DNA extracted by a low-toxic and effective method is free of contamination and degradation.Through the detection by the nucleic acid protein analyzer,the DNA concentration,yield,A260/A280 and A260/A230 of 5 peanut varieties were 419.6-498.2 ng/μL,20.98-24.91μg/g,1.89-1.96 and 2.03-2.28,respectively.The DNA was of high quality and can be completely digested by EcoRI restriction enzymes,and also can be used for SCoT and SRAP molecular marker technology analysis.The RNA extracted from different tissues of peanuts showed no visible DNA bands by non-denaturing agarose gel electrophoresis.The separated 28S bands were brighter than 18S.The ratio of A260/A280 and A260/A230 showed that the RNA quality was good and can be used for reverse transcription,RT-PCR of actin gene and amplification of cDNA-SCoT gene differential display technique.[Conclusions]This experiment established a low-toxic and effective method for extracting DNA and total RNA from peanuts.Compared with traditional methods,this method is more time-saving and cheaper than commercial kits.The most important point is that this method does not use toxic reagents such as phenol,chloroform and isopropanol.Thus,it is expected to be widely applied in molecular biology research.展开更多
基金Projects of National Natural Science Foundation of China(3166042831960409+2 种基金31960416)Projects of Guangxi Natural Science Foundation of China(2018GXNSFDA281027,2018GXNSFDA294004,2017GXNSFAA198032)Science and Technology Development Fund Project of Guangxi Academy of Agricultural Sciences(Gui Nong Ke 2018YM06,Gui Nong Ke 2017JZ13,31960409,Gui Nong Ke 2018YT12).
文摘[Objectives]To establish a non-toxic and efficient method for extracting DNA and total RNA from peanuts and laying a solid foundation for the molecular biology study of peanuts.[Methods]Based on the principle and method of purifying nucleic acids by silica gel adsorption at high salt and low pH condition,a non-toxic and efficient method to extract peanut DNA and total RNA using cetyltrimethyl ammonium bromide(CTAB)extraction solution was designed.The quality and purity of nucleic acids were detected by agarose gel electrophoresis and nucleic acids protein analyzer,respectively.The quality of DNA was further verified by enzyme digestion and PCR amplification using molecular marker techniques.The quality of total RNA was further verified by reverse transcription(RT)-PCR of actin gene and cDNA-SCoT gene differential display technique.[Results]The agarose gel electrophoresis test showed that the peanut DNA extracted by a low-toxic and effective method is free of contamination and degradation.Through the detection by the nucleic acid protein analyzer,the DNA concentration,yield,A260/A280 and A260/A230 of 5 peanut varieties were 419.6-498.2 ng/μL,20.98-24.91μg/g,1.89-1.96 and 2.03-2.28,respectively.The DNA was of high quality and can be completely digested by EcoRI restriction enzymes,and also can be used for SCoT and SRAP molecular marker technology analysis.The RNA extracted from different tissues of peanuts showed no visible DNA bands by non-denaturing agarose gel electrophoresis.The separated 28S bands were brighter than 18S.The ratio of A260/A280 and A260/A230 showed that the RNA quality was good and can be used for reverse transcription,RT-PCR of actin gene and amplification of cDNA-SCoT gene differential display technique.[Conclusions]This experiment established a low-toxic and effective method for extracting DNA and total RNA from peanuts.Compared with traditional methods,this method is more time-saving and cheaper than commercial kits.The most important point is that this method does not use toxic reagents such as phenol,chloroform and isopropanol.Thus,it is expected to be widely applied in molecular biology research.
