Establishment Of Easy-DNA Extraction Kit And Its Application In Molecular Identification Of Plant Samples

Deoxyribonucleic acid(DNA)is the genetic material of most living organisms.Gene detection technology based on DNA separation,purification and amplification is the most important means of biological identification,widely used in biological germplasm identification,prevention of animal and plant diseases,food safety monitoring,genetically modified organisms detection and other fields.DNA purification method based on hexadecyltrimethylammonium bromide(CTAB)has been considered as the standard protocol for DNA extraction.But the complex and time-consuming procedures can't meet the needs of rapid molecular identification.The method of using cellulose filter paper strips to transfer the DNA in the biological tissue lysate to the nucleic acid amplification system shortened the DNA extraction time to 30 seconds.Therefore,cellulose filter paper is getting more focus in rapid DNA extraction method.The application of cellulose filter paper is mainly reported in the technology of rapid detection of transgenic animals or pathogenic bacteria,but there are few studies on the rapid extraction of DNA from plants.Thus,further optimizing the method of extracting DNA from plant samples using cellulose filter paper will help promoting the development of basic research in biological laboratory,identification of plant germplasm resources,identification of transgenic plants,food and medicinal materials and other molecular identification technologies.In the study,the published DNA extraction method based on cellulose filter paper was optimized in the following aspects.Firstly,to overcome the shortcoming of deformation of the filter paper strips and thus taking away too much nucleic acid amplification reaction fluid,the original filter paper strip was modified by sticking the filter paper on a polyvinyl chloride resin(PVC)sheet.This modified method was named EZ-D,for easy DNA extraction.The optimized filter paper strip was designated as EZ-D stick.DNA captured by EZ-D stick showed higher PCR efficiency than the DNA extracted by the published filter paper strip due to the more stable performance of the EZ-D stick.In addition,the optimized DNA extraction buffer exhibited higher DNA extraction efficiency and satisfied the rapid extraction of most plant samples.When compared with hexadecyltrimethylammonium bromide(CTAB)DNA extraction method,the EZ-D method showed the advantages of faster,easier,and cheaper.EZ-D could not only shorten the DNA extraction time from three hours to 30 seconds,but also reduced the cost of DNA extraction from 0.9 to 0.2 CNY per sample.At the same time,EZ-D method made DNA extraction technology free from the constraints of instruments and was more suitable for on-site molecular detection.The ability of EZ-D to extract DNA from plant samples and the amplifiability of the obtained DNA were evaluated in this study.EZ-D was successfully used for DNA extraction from a variety of plant species and plant tissues.A regular PCR reaction can detect the EZ-D-extracted DNA template at concentration as low as 0.1 ng/?L.Evaluation of the EZ-D showed that DNA extracts could be successfully amplified by PCR reaction for DNA fragments up to 3000 bp in length and up to 80% in GC content.Moreover,when EZ-D was combined with the loop-mediated isothermal amplification(LAMP)method,DNA identification of biological samples could be achieved without the need for specialized equipment.In conclusion,this study has developed an optimized DNA purification method EZD based on the published protocol.EZ-D shows great advantages in application and can be used widely in laboratories where equipment is limited and rapid results are required.EZ-D not only enrich the research about the plant DNA purification protocol using cellulose filter paper,but contributes to the popularization of cellulose filter paper in quick plant DNA purification.