Development and forensic validation of human genomic DNA quantification kit

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DNA quantification is an essential step for successful multiplex short tandem repeat (STR) polymerase chain reactions (PCR), which are used for confirming identities using human genomic DNA. The new DNA quantification kit, named the National Forensic Service Quantification (NFSQ) kit, simultaneously provides total human DNA concentration, human male DNA concentration, and a DNA degradation index (DI) using multiplex TaqMan fluorescent probes. The NFSQ was validated according to developmental validation guidelines from the SWGDAM and MIQE. NFSQ detected up to 0.00128 ng/μL and could detect male DNA up to a 1:8000 ratio of male to female DNA. In PCR inhibitor tests, NFSQ could measure DNA at a concentration of 200 ng/μL of humic acid and 600 μM of hematin. The NFSQ kit showed a DI value trend similar to other qPCR kits. In the reproducibility study, the coefficient of variation of the NFSQ kit was within 10%. The quantitative results of the casework samples obtained using the NFSQ kit were consistent with the STR interpretation results. The NFSQ kit can be useful in the human identification process, as it has detection capabilities similar to those of other comparable quantification kits.

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  • 26 March 2020

    The above article was published with two author names being incorrect. The published paper states ������Hyeun Kyu Yoon and Ki min Seong���, whereas it should be ������Hyun Kyu Yoon and Ki Min Seong���.


  1. 1.

    Butler JM (2011) Advanced topics in forensic DNA typing: methodology. National Institute of standards and Technology, Gaithersburg

    Google Scholar 

  2. 2.

    Kitayama T, Fujii K, Nakahara H, Mizuno N, Kasai K, Yonezawa N, Sekiguchi K (2013) Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR. Legal Med 15:1–6

    CAS  Article  Google Scholar 

  3. 3.

    Ambers A, Turnbough M, Benjamin R, Gill-King H, King J, Sajantila A, Budowle B (2016) Modified DOP-PCR for improved STR typing of degraded DNA from human skeletal remains and bloodstains. Legal Med 18:7–12

    CAS  Article  Google Scholar 

  4. 4.

    Butler JM (2006) Genetics and genomics of core short tandem repeat loci used in human identity. J Forensic Sci 51(2):253–265

    CAS  Article  Google Scholar 

  5. 5.

    Kline MC, Duewer DL, Redman JW, Butler JM (2003) Mixed stain study 3: DNA quantitation accuracy and its influence on short tandem repeat multiplex signal intensity. Anal Chem 46(5):1199–1210

    Google Scholar 

  6. 6.

    Lee SB, McCord B, Buel E (2014) Advances in forensic DNA quantification: a review. Electrophoresis 35(21–22):3044–3052

    CAS  Article  Google Scholar 

  7. 7.

    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

    Article  Google Scholar 

  8. 8.

    Deagle BE, Eveson JP, Buel E (2006) Quantification of damage in DNA recovered from highly degraded samples-a case study on DNA in faeces. Front Zool 3:11

    Article  Google Scholar 

  9. 9.

    Alonso A, Martin P, Albarran C, Garcia P, Garcia O, Fernandez de Simon L, Garcia-Hirschfeld J, Sancho M, de la Rua C, Fernandez-Piqueras J (2004) Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies. Forensic Sci Int 139:141–149

    CAS  Article  Google Scholar 

  10. 10.

    Van Guilder HD, Vrana KE, Freeman WM (2008) Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 44(5):619–626

    Article  Google Scholar 

  11. 11.

    Udvardi MK, Czechowski T, Scheible WR (2008) Eleven Golden rules of quantitative RT-PCR. Plant Cell 20(7):1736–1737

    CAS  Article  Google Scholar 

  12. 12.

    Validation Guidelines for DNA Analysis Methods – Scientific Working Group on DNA analysis Methods. Accessed 18 December 2018

  13. 13.

    Lin SW, Li C, Ip SCY (2018) A performance study on three qPCR quantification kits and their compatibilities with the 6-dye DNA profiling systems. Forensic Sci Int Genet 33:72–83

    Article  Google Scholar 

  14. 14.

    Stephen AB, Vladimir B, Jeremy AG, Jan H, Jim H, Mikael K, Reinhold M, Tania N, Michael WP, Gregory LS, Jo V (2009) Carl TW (2009) the MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611–622

    Article  Google Scholar 

  15. 15.

    McMichael GL, Highet AR (2011) Comparison of DNA extraction methods from small samples of newborn screening cards suitable for retrospective perinatal viral research. J Biomol Tech 22(1):5–9

    PubMed  PubMed Central  Google Scholar 

  16. 16.

    QIAamp DNA Micro Handbook Accessed 18 December 2018

  17. 17.

    Girish PS, Anjaneyulu AS, Viswas KN, Anand M, Rajkumar N, Shivakumar BM, Bhaskar S (2004) Sequence analysis of mitochondrial 12S rRNA gene can identify meat species. Meat Sci 66(3):551–556

    CAS  Article  Google Scholar 

  18. 18.

    Swango KL, Timken MD, Date Chong M, Buoncristiani MR (2006) A quantitative PCR assay for the assessment of DNA degradation in forensic samples. Forensic Sci Int 158:14–26

    CAS  Article  Google Scholar 

  19. 19.

    Elsner HI, Lindblad EB (1989) Ultrasonic degradation of DNA. DNA 8(10):697–701

    CAS  Article  Google Scholar 

  20. 20.

    Swango KL, Hudlow WR, Timken MD, Buoristiani MR (2007) Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples. Forensic Sci Int 170:35–45

    CAS  Article  Google Scholar 

  21. 21.

    Holt A, Wootton SC, Mulero JJ, Brzoska PM, Langit E, Green RL (2016) Developmental validation of the Quantifiler® HP and trio kits for human DNA quantification in forensic samples. Forensic Sci Int Genet 21:145–157

    CAS  Article  Google Scholar 

  22. 22.

    Maura Barbisin, Rixun Fang, Cristin E. O’Shea, Lisa M. Calandro, Monohar R. Furtado, J.G. Shewale (2009) Developmental validation of the Quantifiler® duo DNA quantification kit for simultaneous quantification of total human and human male DNA and detection of PCR inhibitors in biological samples. J Forensic Sci 54305–319, 54, 305

  23. 23.

    Quantifiler® HP and Trio DNA Quantification kits User Guide, Accessed 18 December 2018

  24. 24.

    PowerQuant™ System Technical Manual, Accessed 18 December 2018

  25. 25.

    Investigator® Quantiplex pro kit Handbook, Accessed 18 December 2018

  26. 26.

    Schaiff WT, Hruska KA Jr, McCourt DW, Green M, Schwartz BD (1992) HLA-DR associates with specific stress proteins and is retained in the endoplasmic reticulum in invariant chain negative cells. J Exp Med 176(3):657–666

    CAS  Article  Google Scholar 

  27. 27.

    Jonsson AK, Rask L (1989) Human class II DNA and DOB genes display low sequence variability. Immunogenetics 29(6):411–413

    CAS  Article  Google Scholar 

  28. 28.

    Bioanalytical Method Validation Guidance for Industry May 2018,, Accessed 18 May 2019

  29. 29.

    Holmes AS, Houston R, Elwick K, Gangitano D, Hughes-Stamm S (2017) Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples. Int J Legal Med 129:1–11

    Google Scholar 

  30. 30.

    Karni M, Zidon D, Polak P, Zalevsky Z, Shefi O (2013) Thermal degradation of DNA. DNA Cell Biol 32:298–301

    CAS  Article  Google Scholar 

  31. 31.

    Cho Y, Kim HS, Kim MH, Park M, Kwon H, Lee YH, Lee DS (2018) Validation of reduced reagent volumes in the implementation of the Quantifiler® trio DNA quantification kit. J Forensic Sci 63(2):517–525

    CAS  Article  Google Scholar 

  32. 32.

    Goecker ZC, Swiontek SE, Lakhtakia A, Roy R (2016) Comparison of Quantifiler® trio and InnoQuant™ human DNA quantification kits for detection of DNA degradation in developed and aged fingerprints. Forensic Sci Int 263:132–138

    CAS  Article  Google Scholar 

  33. 33.

    Ewing MM, Thompson JM, McLaren RS, Purpero VM, Thomas KJ, Dobrowski PA, DeGroot GA, Romsos EL, Storts DR (2016) Human DNA quantification and sample quality assessment: developmental validation of the PowerQuant® system. Forensic Sci Int Genet 23:166–177

    CAS  Article  Google Scholar 

  34. 34.

    Vranes M, Scherer M, Elliott K (2017) Development and validation of the Investigator® Quantiplex Pro kit for qPCR-based examination of the quantity and quality of human DNA in forensic samples. Forensic Sci Int Genet Suppl Ser 6:e518–e519

    Article  Google Scholar 

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This work was supported by the Korean government and by a grant (NFS2019DNA02) from the Forensic Research program of the National Forensic Service (NFS).

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Correspondence to Si-Keun Lim.

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The authors declare that they have no conflict of interest.

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All procedures performed in studies involving human participants were in accordance with the ethical standards by Institutional Review Board (IRB) of National Forensic Service (Approval No. 906-170118-BR-003-02).

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Kim, J., Jung, J.Y., Kwon, S.Y. et al. Development and forensic validation of human genomic DNA quantification kit. Int J Legal Med 134, 963–975 (2020).

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  • DNA quantification
  • Forensic science
  • Real-time PCR
  • Short tandem repeat