Abstract
Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194–0.303 ng/μl at 0–1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003–0.168 ng/μl at 2.4–4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The C T values of an internal PCR control (IPC) were 28.0–31.0, 36.5–37.1, and undetermined at 0–1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high C T values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler™ kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA.
References
Applied Biosystems (2006) AmpFℓSTR® Identifiler® PCR Amplification Kit User’s Manual. Applied Biosystems, Foster City
Promega Corporation (2008) PowerPlex® 16 System Technical Manual. Promega, Madison
Butler JM (2005) Forensic DNA typing: biology, technology, and genetics of STR markers, 2nd edn. Elsevier, Burlington
Budowle B, Eisenberg AJ, van Daal A (2009) Validity of low copy number typing and applications to forensic science. Croat Med J 50:207–217
Caragine T, Mikulasovich R, Tamariz J, Bajda E, Sebestyen J, Baum H, Prinz M (2009) Validation of testing and interpretation protocols for low template DNA samples using AmpFlSTR Identifiler. Croat Med J 50:250–267
Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W, Morling N (2008) Comparison of five DNA quantification methods. Forensic Sci Int Genet 2:226–230
Nicklas JA, Buel E (2003) Quantification of DNA in forensic samples. Anal Bioanal Chem 376:1160–1167
Sutlović D, Definis Gojanović M, Andelinović S, Gugić D, Primorac D (2005) Taq polymerase reverses inhibition of quantitative real time polymerase chain reaction by humic acid. Croat Med J 46:556–562
Definis Gojanović M, Sutlović D (2007) Skeletal remains from World War II mass grave: from discovery to identification. Croat Med J 48:520–527
Keyser C, Bouakaze C, Crubézy E, Nikolaev VG, Montagnon D, Reis T, Ludes B (2009) Ancient DNA provides new insights into the history of south Siberian Kurgan people. Hum Genet 126:395–410
Vanek D, Saskova L, Koch H (2009) Kinship and Y-chromosome analysis of 7th century human remains: novel DNA extraction and typing procedure for ancient material. Croat Med J 50:286–295
Seo SB, Zhang A, Kim HY, Yi JA, Lee HY, Shin DH, Lee SD (2010) Technical note: Efficiency of total demineralization and ion-exchange column for DNA extraction from bone. Am J Phys Anthropol 141:158–162
Kermekchiev MB, Kirilova LI, Vail EE, Barnes WM (2009) Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples. Nucleic Acids Res 37:e40
Braid MD, Daniels LM, Kitts CL (2003) Removal of PCR inhibitors from soil DNA by chemical flocculation. J Microbiol Methods 52:389–393
Tsai YL, Olson BH (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol 58:2292–2295
Zipper H, Buta C, Lämmle K, Brunner H, Bernhagen J, Vitzthum F (2003) Mechanisms underlying the impact of humic acids on DNA quantification by SYBR Green I and consequences for the analysis of soils and aquatic sediments. Nucleic Acids Res 31:e39
Bachoon DS, Otero E, Hodson RE (2001) Effects of humic substances on fluorometric DNA quantification and DNA hybridization. J Microbiol Methods 47:73–82
Applied Biosystems (2005) Quantifiler™ Human DNA Quantification Kit User’s Manual. Applied Biosystems, Foster City
Mulero JJ, Chang CW, Lagacé RE, Wang DY, Bas JL, McMahon TP, Hennessy LK (2008) Development and validation of the AmpFlSTR MiniFiler PCR Amplification Kit: a MiniSTR multiplex for the analysis of degraded and/or PCR inhibited DNA. J Forensic Sci 53:838–852
Opel KL, Chung D, McCord BR (2010) A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55:25–33
Hawass Z, Gad YZ, Ismail S, Khairat R, Fathalla D, Hasan N, Ahmed A, Elleithy H, Ball M, Gaballah F, Wasef S, Fateen M, Amer H, Gostner P, Selim A, Zink A, Pusch CM (2010) Ancestry and pathology in King Tutankhamun’s family. JAMA 303:638–647
Gamba C, Fernández E, Tirado M, Pastor F, Arroyo-Pardo E (2011) Brief communication: Ancient nuclear DNA and kinship analysis: the case of a medieval burial in San Esteban Church in Cuellar (Segovia, Central Spain). Am J Phys Anthropol 144:485–491
Applied Biosystems (2007) AmpFℓSTR® MiniFiler™ PCR Amplification Kit User Guide. Applied Biosystems, Foster City
Ricci U, Marchi C, Previderè C, Fattorini P (2006) Quantification of human DNA by real time PCR in forensic casework. Int Congr Ser 1288:750–752
Acknowledgements
This work was supported by grant no 04-2010-0500 from the Seoul National University Hospital (SNUH) Research Fund and by Future-based Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No. 2010-0020631).
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Seo, S.B., Lee, H.Y., Zhang, A.H. et al. Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency. Int J Legal Med 126, 961–968 (2012). https://doi.org/10.1007/s00414-011-0616-z
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DOI: https://doi.org/10.1007/s00414-011-0616-z