In-silico prediction of PCR inhibition mechanism and exploitation of bacterial EPS mediated calcium nanoparticles as PCR facilitator for efficient forensic DNA analysis

Abstract

The routine Forensic DNA analysis result is strongly influenced by the mitigation strategy employed against potential PCR inhibitors. Bacterial Extracellular Polymeric Substances (EPS) mediated Calcium Nanoparticle and EPS combination was explored to mitigate the routine DNA extraction chemicals for a successful DNA profile generation. In-silico analysis predicted Proteinase-K (−871. 2 kCal/mol) to affect Taq DNA Polymerase most adversely, followed by Chloroform (−5.2 kCal/ mol), Phenol (−4.5 kCal/ mol), SDS (−4.1 kCal/ mol), Ethanol (−3.2 kCal/ mol), and EDTA (−2.6 kCal/ mol). 150–200 nm of needle-shaped Ca NPs and EPS (1 mg/ml) combination showed a significant increase in the PCR process influenced by the inhibitor compounds tested. The EPS + Ca NPs formulation showed the amplification efficiency of the CYCLO gene at 1.61–4.02 times higher than the inhibitor compounds. Besides, this formulation was able to amplify the 23 autosomal STR markers simultaneously in a multiplex PCR in the presence of the inhibitor compounds. The formulation was able to mitigate the chemical contaminants and was able to generate a complete profile with a high inter-locus balance. Thus, the EPS + Ca NPs formulation is deemed to be of suitable use in mitigating the routinely used DNA extraction chemical contaminations of DNA in analyzing forensic biological samples.