Plant genomic DNA extraction for selected herbs and sequencing their internal transcribed spacer regions amplified by specific primers

Abstract

This study was focused on plant genomic DNA extraction and sequencing from five commonly used medicinal herbs, namely Impatiens balsamina, Ficus deltoidea, Centella asiatica, Andrographis paniculata and Orthosiphon aristatus. This molecular technique is another highly reliable alternative for plant species identification besides phytochemical profiling. Three cetyl hexadecyltrimethylammonium bromide (CTAB) based methods with slight modification on incubation time, salt content and other additives were used for DNA extraction. The CTAB method of Doyle and Doyle produced higher DNA concentration from I. balsamina, most probably due to the presence of ammonium acetate in the washing buffer and longer incubation time (2 h). The CTAB based method was suitable for A. paniculata because a high DNA concentration of acceptable quality was obtained for all the modified methods. However, O. aristatus was likely to have a lower DNA concentration (33-87 μg/g) and quality, probably due to the high concentration of phenolic compounds, particularly rosmarinic acid. The extracted genomic DNA was effectively amplified by a polymerase chain reaction using a universal primer of internal transcribed spacer (ITS), particularly AB101 and AB102 at the optimum annealing temperature of 48oC. The DNA sequences were analyzed by phenetic analysis and it was found that they have high similarity with the nucleotide sequences of ITS regions for similar plant species in the GenBank database of the National Center for Biotechnology Information.