Optimization of primer and polymerase chain reaction conditions to amplify COI locus for identification of Purnajiwa (Euchresta horsfieldii (Lesch.) Benn.) collected from Bedugul, Bali

Abstract

The polymerase chain reaction (PCR) has been used for molecular research to amplify DNA fragments, especially from a small amount of genetic material. PCR has been applied for numerous researches, including for plant molecular identification. A good PCR product is dependent on good amplification of the DNA segment in optimum condition. This research was conducted to optimize the primer combination, and the annealing temperature for DNA barcoding application of Balinese rare medicinal plant (Euchresta horsfieldii (Lesch.) Benn.) collected from Bedugul. COI is a suitable primer to identify unknown species to higher taxa levels and species with high phenotypic plasticity. The range of annealing temperatures was tested to amplify the combination of five COI primers: GWSF and GWSF5 for forwarding primers and GWSR, GWSR3, and GWSR5 for reverse primers. The annealing temperature was 52, 54, 56, 58, 60°C for 30 seconds. The result showed that GWSF-GWSR, GWSF-GWSR5, GWSF5-GWSR, GWSF5-GWSR5, and GWSF5-GWSR3 produced multiple bands, the double band that cannot be cut, multiple bands, faint amplification band for GWSF5-GWSR5 and GWSF5-GWSR3, respectively. Primer combination of GWSF-GWSR3 with an annealing temperature of 52°Cproduced double bands that were available to have proceeded for sequencing. In conclusion, the combination of GWSF-GWSR3 that is annealed at 52°C produced the best amplification band. The primer combinations and conditions can be used for further identification of Purnajiwa collected from Bedugul.