International Journal of Legal Medicine

, Volume 126, Issue 6, pp 961–968 | Cite as

Effects of humic acid on DNA quantification with Quantifiler® Human DNA Quantification kit and short tandem repeat amplification efficiency

  • Seung Bum Seo
  • Hye Young Lee
  • Ai Hua Zhang
  • Hye Yeon Kim
  • Dong Hoon Shin
  • Soong Deok Lee
Technical Note


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.


DNA quantification Real-time PCR Short tandem repeat Humic acid PCR inhibitor 



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).


  1. 1.
    Applied Biosystems (2006) AmpFSTR® Identifiler® PCR Amplification Kit User’s Manual. Applied Biosystems, Foster CityGoogle Scholar
  2. 2.
    Promega Corporation (2008) PowerPlex® 16 System Technical Manual. Promega, MadisonGoogle Scholar
  3. 3.
    Butler JM (2005) Forensic DNA typing: biology, technology, and genetics of STR markers, 2nd edn. Elsevier, BurlingtonGoogle Scholar
  4. 4.
    Budowle B, Eisenberg AJ, van Daal A (2009) Validity of low copy number typing and applications to forensic science. Croat Med J 50:207–217PubMedCrossRefGoogle Scholar
  5. 5.
    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 50:250–267PubMedCrossRefGoogle Scholar
  6. 6.
    Nielsen K, Mogensen HS, Hedman J, Niederstätter H, Parson W, Morling N (2008) Comparison of five DNA quantification methods. Forensic Sci Int Genet 2:226–230PubMedCrossRefGoogle Scholar
  7. 7.
    Nicklas JA, Buel E (2003) Quantification of DNA in forensic samples. Anal Bioanal Chem 376:1160–1167PubMedCrossRefGoogle Scholar
  8. 8.
    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 46:556–562PubMedGoogle Scholar
  9. 9.
    Definis Gojanović M, Sutlović D (2007) Skeletal remains from World War II mass grave: from discovery to identification. Croat Med J 48:520–527PubMedGoogle Scholar
  10. 10.
    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 126:395–410PubMedCrossRefGoogle Scholar
  11. 11.
    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 50:286–295PubMedCrossRefGoogle Scholar
  12. 12.
    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 141:158–162PubMedGoogle Scholar
  13. 13.
    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:e40PubMedCrossRefGoogle Scholar
  14. 14.
    Braid MD, Daniels LM, Kitts CL (2003) Removal of PCR inhibitors from soil DNA by chemical flocculation. J Microbiol Methods 52:389–393PubMedCrossRefGoogle Scholar
  15. 15.
    Tsai YL, Olson BH (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction. Appl Environ Microbiol 58:2292–2295PubMedGoogle Scholar
  16. 16.
    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:e39PubMedCrossRefGoogle Scholar
  17. 17.
    Bachoon DS, Otero E, Hodson RE (2001) Effects of humic substances on fluorometric DNA quantification and DNA hybridization. J Microbiol Methods 47:73–82PubMedCrossRefGoogle Scholar
  18. 18.
    Applied Biosystems (2005) Quantifiler™ Human DNA Quantification Kit User’s Manual. Applied Biosystems, Foster CityGoogle Scholar
  19. 19.
    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 53:838–852PubMedCrossRefGoogle Scholar
  20. 20.
    Opel KL, Chung D, McCord BR (2010) A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 55:25–33PubMedCrossRefGoogle Scholar
  21. 21.
    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 303:638–647PubMedCrossRefGoogle Scholar
  22. 22.
    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 144:485–491PubMedCrossRefGoogle Scholar
  23. 23.
    Applied Biosystems (2007) AmpFSTR® MiniFiler™ PCR Amplification Kit User Guide. Applied Biosystems, Foster CityGoogle Scholar
  24. 24.
    Ricci U, Marchi C, Previderè C, Fattorini P (2006) Quantification of human DNA by real time PCR in forensic casework. Int Congr Ser 1288:750–752CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Seung Bum Seo
    • 1
  • Hye Young Lee
    • 1
  • Ai Hua Zhang
    • 1
  • Hye Yeon Kim
    • 2
  • Dong Hoon Shin
    • 3
    • 4
  • Soong Deok Lee
    • 1
    • 4
  1. 1.Department of Forensic Medicine, College of MedicineSeoul National UniversitySeoulSouth Korea
  2. 2.DNA Analysis Laboratory, Division of Forensic ScienceSupreme Public Prosecutor’s OfficeSeoulSouth Korea
  3. 3.Department of Anatomy, College of MedicineSeoul National UniversitySeoulSouth Korea
  4. 4.Institute of Forensic Medicine, College of MedicineSeoul National UniversitySeoulSouth Korea

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