The role of activated carbon in elimination of polymerase chain reaction inhibitors in forensic biological samples

Forenzični biološki uzorci često sadržavaju male količine DNA uz prisustvo smjese inhibitora umnažanja DNA. Tijekom izolacije DNA s podloge dijelovi podloge kao potencijalni inhibitori mogu se izolirati zajedno s genomskom DNA, čime se smanjuje ili u potpuno onemogućuje umnažanje mikrosatelitnih lokusa. Cilj ovog istraživanja jest ukloniti inhibitore umnažanja DNA pomoću aktivnog ugljena uz što manji gubitak humane DNA. Kvaliteta izolirane DNA testirana je umnažanjem deset mikrosatelitnih lokusa. Uspoređena je efikasnost neafinitetnih metoda izolacije genomske DNA (Chelex i organska izolacija), kao i metoda pročišćavanja ultrafiltracijom (Amicon komercijalna kolona) i adsorpcijom (aktivni ugljen). Općenito, Chelex se pokazao kao učinkovitija metoda izolacije DNA, a usporedbom metoda pročišćavanja nije bilo statistički značajne razlike u kvaliteti umnožene DNA. Primjena aktivnog ugljena pokazala se jednostavnom, brzom, ekonomičnom i reproducibilnom metodom pročišćavanja. Aktivni ugljen uspješno je adsorbirao hematin, prirodni inhibitor iz krvi, bez značajnih gubitaka DNA. Nadalje, smanjena je koncentracija inhibitora iz podloge kao što su humusne tvari iz zemlje i tanini iz drva. Amicon se pokazao kao efikasnija metoda za uklanjanje indiga iz trapera kao podloge. Pročišćavanje bioloških uzoraka temeljeno na fizikalnim i kemijskim svojstvima aktivnog ugljena dosad nije provedeno, stoga spoznaje utvrđene u ovom istraživanju daju prvi uvid u novu primjenu tog dobro poznatog adsorbensa.Forensic biological samples often contain small quantities of DNA in presence of mixture of DNA amplification inhibitors. During the genomic DNA extraction, parts of the substrate, as potential inhibitors, could be co-extracted with genomic DNA and reduce or completely disable amplification of microsatellite loci. Aim of this study is to eliminate inhibitors of DNA amplification using activated carbon, without significant loss of human DNA. Quality of extracted DNA was tested by amplification of ten microsatellite loci. In this doctoral thesis, efficiency of non-affinity DNA extraction methods (Chelex and organic extraction) as well as purification methods based on ultrafiltration (Amicon commercial column) and adsorption (activated carbon) were compared. Generally, Chelex extraction method was more efficient, while no statistically significant difference was obtained if purification methods were compared. Application of activated carbon was shown as simple, fast, economic and reproducible purification method. Activated carbon effectively adsorbed hematin, as inhibitor naturally originated from blood, without significant loss of DNA. In addition, concentration of inhibitors present in the substrate, such as humic substances in soil substrate, as well as tannins in wood, was reduced. Amicon was more efficient method for elimination of indigo dye as inhibitor present in denim substrate. Since purification of biological samples based solely on chemical and physical characteristics of activated carbon has not yet been performed, results of this research provide the first insight into a new application of this well known adsobent