Advertisement

Comparison of DNA polymerases for improved forensic analysis of challenging samples

      Highlights

      • An efficient enzyme is crucial in forensic DNA analysis if inhibitors are present.
      • KAPA3G Plant and KAPA2G Robust enzymes have high resistance to certain inhibitors.
      • The KAPA polymerases counteract inhibitors in analysis of challenging samples.

      Abstract

      Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples.

      Keywords

      To read this article in full you will need to make a payment

      Purchase one-time access:

      Academic & Personal: 24 hour online accessCorporate R&D Professionals: 24 hour online access
      One-time access price info
      • For academic or personal research use, select 'Academic and Personal'
      • For corporate R&D use, select 'Corporate R&D Professionals'

      Subscribe:

      Subscribe to Forensic Science International: Genetics
      Already a print subscriber? Claim online access
      Already an online subscriber? Sign in
      Institutional Access: Sign in to ScienceDirect

      References

        • Schrader C.
        • Schielke A.
        • Ellerbroek L.
        • Johne R.
        PCR inhibitors – occurrence, properties and removal.
        J. Appl. Microbiol. 2012; 113: 1014-1026https://doi.org/10.1111/j.1365-2672.2012.05384.x
        • Eckhart L.
        • Bach J.
        • Ban J.
        • Tschachler E.
        Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity.
        Biochem. Biophys. Res. Commun. 2000; 271: 726-730https://doi.org/10.1006/bbrc.2000.2716
        • Hu Q.
        • Liu Y.
        • Yi S.
        • Huang D.
        A comparison of four methods for PCR inhibitor removal.
        Forensic Sci. Int. Genet. 2015; 16: 94-97https://doi.org/10.1016/j.fsigen.2014.12.001
        • Alaeddini R.
        Forensic implications of PCR inhibition – a review.
        Forensic Sci. Int. Genet. 2012; 6: 297-305https://doi.org/10.1016/j.fsigen.2011.08.006
        • Monroe C.
        • Grier C.
        • Kemp B.M.
        Evaluating the efficacy of various thermo-stable polymerases against co-extracted PCR inhibitors in ancient DNA samples.
        Forensic Sci. Int. 2013; 228: 142-153https://doi.org/10.1016/j.forsciint.2013.02.029
        • Kreader C.A.
        Relief of amplification inhibition in PCR with bovine serum albumin or T4 gene 32 protein.
        Appl. Environ. Microbiol. 1996; 62: 1102-1106
        • Terpe K.
        Overview of thermostable DNA polymerases for classical PCR applications: from molecular and biochemical fundamentals to commercial systems.
        Appl. Microbiol. Biotechnol. 2013; 97: 10243-10254https://doi.org/10.1007/s00253-013-5290-2
        • Abu Al-Soud W.
        • Radstrom P.
        Capacity of nine thermostable DNA polymerases To mediate DNA amplification in the presence of PCR-inhibiting samples.
        Appl. Environ. Microbiol. 1998; 64: 3748-3753
        • Eilert K.D.
        • Foran D.R.
        Polymerase resistance to polymerase chain reaction inhibitors in bone*.
        J. Forensic Sci. 2009; 54: 1001-1007https://doi.org/10.1111/j.1556-4029.2009.01116.x
        • Schori M.
        • Appel M.
        • Kitko A.
        • Showalter A.M.
        Engineered DNA polymerase improves PCR results for plastid DNA.
        Appl. Plant Sci. 2013; 1https://doi.org/10.3732/apps.1200519
        • Budowle B.
        • Wilson M.R.
        • DiZinno J.A.
        • Stauffer C.
        • Fasano M.A.
        • Holland M.M.
        • Monson K.L.
        Mitochondrial DNA regions HVI and HVII population data.
        Forensic Sci. Int. 1999; 103: 23-35https://doi.org/10.1016/S0379-0738(99)00042-0
        • Gettings K.B.
        • Kiesler K.M.
        • Vallone P.M.
        Performance of a next generation sequencing SNP assay on degraded DNA.
        Forensic Sci. Int. Genet. 2015; 19: 1-9https://doi.org/10.1016/j.fsigen.2015.04.010
        • Andreasson H.
        • Gyllensten U.
        • Allen M.
        Real-time DNA quantification of nuclear and mitochondrial DNA in forensic analysis.
        Biotechniques. 2002; 33 (402–4, 7–11)
        • Nilsson M.
        • Possnert G.
        • Edlund H.
        • Budowle B.
        • Kjellström A.
        • Allen M.
        Analysis of the putative remains of a European patron saint–St. Birgitta.
        PLoS One. 2010; 5: e8986https://doi.org/10.1371/journal.pone.0008986
        • Andrews R.M.
        • Kubacka I.
        • Chinnery P.F.
        • Lightowlers R.N.
        • Turnbull D.M.
        • Howell N.
        Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.
        Nat. Genet. 1999; 23: 147https://doi.org/10.1038/13779
        • Kjellstrom A.
        • Edlund H.
        • Lembring M.
        • Ahlgren V.
        • Allen M.
        An analysis of the alleged skeletal remains of Carin Goring.
        PLoS One. 2012; 7: e44366https://doi.org/10.1371/journal.pone.0044366
        • Maciejewska A.
        • Wlodarczyk R.
        • Pawlowski R.
        The influence of high temperature on the possibility of DNA typing in various human tissues. Folia Histochemica et Cytobiologica/Polish Academy of Sciences.
        Polish Histochem. Cytochem. Soc. 2015; (Nov 6)https://doi.org/10.5603/fhc.a2015.0029