Effect of different DNA extraction methods and DNA dilutions on the amplification success in the PCR of different medicinal and aromatic plants

DNA-based methods to proof identity are becoming increasingly popular. In a routine laboratory it would be desirable to have only as few as possibly different protocols. Therefore we tested the applicability of only one standard polymerase chain reaction (PCR) protocol in nine different plant species, five different DNA extraction methods and three DNA dilutions. DNA was extracted from roots and rhizomes of Valeriana officinalis, roots of Panax ginseng, leaves of Mentha x piperita and Salvia officinalis, flower heads of Matricaria chamomilla, (pseudo-)fruits of Foeniculum vulgare and Juniperus communis and bark of Salix sp. and Cinnamomum verum. PCR conditions were kept constant. Three extraction methods were commercial DNA extraction kits (Nucleospin (R) Plant (Macherey-Nagel), DNeasy (R) Plant Mini Kit (Qiagen) and Wizard (R) Genomic DNA Purification Kit (Promega)), two methods were based on CTAB. The amount of extracted DNA was measured with a fluorimetric method and the applicability was tested by amplifying two DNA loci that became standard in molecular taxonomy with standard primers, the plastid psbA-trnH intergenic spacer and the nuclear internal transcribed spacer region (ITS). The results showed that the DNA concentration used in PCR should be kept variable and that the concentration is in dependency on the DNA amount and quality as well as the amount of secondary compounds remaining in the DNA extract. The quality of the sample material had a higher influence on the PCR amplification success than the choice of the extraction method, which was between 67% and 100% depending on the extraction method and the amplified locus. Furthermore, shorter amplification products showed a higher success rate than longer amplification products.