Application of PCR-RFLP and MASA analyses on 18S ribosomal RNA gene sequence for the identification of three Ginseng drugs.

Abstract

In order to develop convenient and reproducible methods for the identification of Ginseng drugs at a DNA level, PCR-Restriction fragment length polymorphism (PCR-RFLP) and Mutant allele specific amplification (MASA) analyses were applied, based on differences of the 18S rRNA gene sequence among three Panax species. The PCR product of each species on the 18S rRNA gene was digested with the restriction enzymes Ban II and Dde I. Each fragment gave unique electrophoretic profiles for each species (PCR-RFLP analysis). The extracted DNA of each species was amplified by PCR using a designed species-specific oligonucleotide primer. The expected size of the fragments corresponding to each species were detected only when the optimum temperature and reaction time for annealing and extension were established (MASA analysis). These two analytical methods were carried out on three Ginseng drugs and the same results as in their original plants were obtained. The results suggest that PCR-RFLP and MASA analyses under the established conditions are convenient for identifying three Ginseng drugs. Moreover, to insure completion of the identification, a partial sequence of the plastid gene matK was determined in addition to the 18S rRNA gene. The gene sequences of three Panax species were of 1259 base pairs and that of P. quinquefolius was different from the other two at nucleotide position 102.