Optimized DNA extraction and purification method from Alchemilla species using polyethylene glycol

Pure DNA is essential in various techniques of molecular biology and its extraction from plants to produce large amounts of secondary metabolites is a difficult task. Alchemilla is known to synthesize a large number of secondary metabolites which reduce the quality of the extracted DNA. This study, aimed to set up a method for high-quality DNA isolation from Alchemilla leaf. For this purpose, three extraction methods were examined and a comparison concerning price, simplicity, and security was carried out. We also optimized a CTAB-based method using increasing the volume and concentration of CTAB buffer, lysis time, and cold incubation period, performing six times dilutions, and three times precipitations, adding polyethylene glycol, and removing toxic or expensive materials. The results showed that, ۲۶۰/۲۸۰ and ۲۶۰/۲۳۰ ratios of extracted DNA by the optimized method with the concentration of ۵۹۵–۳۸۷ ng/µL were ۱.۷۵–۱.۸۲ and ۱.۵۶–۱.۶۸, respectively. The quality of extracted DNA by this method was significantly higher (p < ۰.۰۰۱) than that of other ways, so that all samples were positive for DNA, as assessed by electrophoresis and PCR. The optimized method was simple, effective, reproducible, relatively non-toxic, and inexpensive. The results revealed that, this method was successful in producing large amounts of high-quality amplifiable DNA.

Keywords:

Nucleic acid purity , Phenolic compounds , polysaccharide compounds , Secondary metabolites , spectrophotometry