TY - JOUR
T1 - The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit
AU - Combs, Laura Gaydosh
AU - Warren, Joseph E.
AU - Huynh, Vivian
AU - Castaneda, Joanna
AU - Golden, Teresa D.
AU - Roby, Rhonda K.
N1 - Funding Information:
Laura Gaydosh Combs is supported by the NIJ Ph.D. Graduate Research Fellowship Program FY 2012, award number 2012-IJ-CX-0017. DNA testing was performed with support from the National Institute of Justice program Using DNA Technology to Identify the Missing, FY 2010, award number 2010-DN-BX-K206. Vivian Huynh is supported by the NIJ Applied Research and Development in Forensic Science for Criminal Justice Purposes program FY 2013, award number 2013-R2-CX-K007. Joanna Castaneda’s work was supported by the National Science Foundation under award number CHE-1004878, as part of an NSF Research Experiences for Undergraduates program in the Department of Chemistry at the University of North Texas. Teresa Golden acknowledges support from the UNT Forensic Science Program. The authors would like to thank Dr. Vallerie H. DeLeon for preparation of metals stock solutions, Alea Ausmer, M.S. and Elizabeth A. Nelson, B.A. for assistance with metal-treated DNA sample preparation, Patricia D. Gonzales, B.F.A. for Adobe ® Photoshop ® CS6 instruction, and Dr. Arthur J. Eisenberg for his continued support.
Publisher Copyright:
© 2015 Elsevier Ireland Ltd. All rights reserved.
PY - 2015/8/3
Y1 - 2015/8/3
N2 - Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler® Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler® Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler® Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that targets human DNA in the Quantifiler® kit. Evidence of inhibition was observed for the human-specific assay at a lower metal concentration than detected by the IPC, for all metals examined except calcium. These results strongly suggest that determination of a "true negative" sample should not be based solely on the failure of the IPC to indicate the presence of a PCR inhibitor and indicate that amplification of all samples should be attempted, regardless of the quantification results.
AB - Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler® Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler® Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler® Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that targets human DNA in the Quantifiler® kit. Evidence of inhibition was observed for the human-specific assay at a lower metal concentration than detected by the IPC, for all metals examined except calcium. These results strongly suggest that determination of a "true negative" sample should not be based solely on the failure of the IPC to indicate the presence of a PCR inhibitor and indicate that amplification of all samples should be attempted, regardless of the quantification results.
KW - 6-Carboxyfluorescein
KW - DNA quantification
KW - Fluorescence spectroscopy
KW - Metal ions
KW - PCR inhibition
UR - http://www.scopus.com/inward/record.url?scp=84938306193&partnerID=8YFLogxK
U2 - 10.1016/j.fsigen.2015.06.013
DO - 10.1016/j.fsigen.2015.06.013
M3 - Article
C2 - 26240969
AN - SCOPUS:84938306193
SN - 1872-4973
VL - 19
SP - 180
EP - 189
JO - Forensic Science International: Genetics
JF - Forensic Science International: Genetics
ER -