A simple improved method for extracting DNA from ethanol-preserved hard ticks and its applications.

Hard tick exoskeletons, composed primarily of chitin, pose a significant challenge for researchers attempting to extract genetic material. This study presents a simple modified, alternative method for extracting DNA from ethanol-preserved hard ticks. The extracted DNA was further used for PCR amplification of phylogenetic markers for population genetics studies. The study also improvises the DNA extraction methods from commercial kits. We have used four DNA extraction methods: Modified Simple Alkaline Lysis, and other commercial kit-based methods (Kit X, Kit Y & Kit Z). The modified method for DNA extraction yielded comparable results in terms of concentration, and purity from all the life stages (adult, nymph, and larvae). The extracted DNA from each method was quantified and subjected to PCR amplification of molecular markers, ITS-1 and ITS-2. The nucleotide sequences from both markers were characterized for the first time and used for phylogenetic analysis of Amblyomma integrum, which is a potential vector for Kyasanur Forest Disease Virus (KFDV), causing monkey fever disease in India. These results demonstrate a cost-effective approach for isolating genomic DNA suitable for PCR amplification and subsequent nucleotide sequencing. Importantly, this simple method offers an option for population genetics study in resource-limited settings, facilitating field research with minimal equipment requirements. Additionally, the study showed tick homogenization can significantly improve DNA yield from commercial kits.