The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit

Laura Gaydosh Combs, Joseph E. Warren, Vivian Huynh, Joanna Castaneda, Teresa D. Golden, Rhonda K. Roby

Research output: Contribution to journalArticleResearchpeer-review

9 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)180-189
Number of pages10
JournalForensic Science International: Genetics
Volume19
DOIs
StatePublished - 3 Aug 2015

Fingerprint

Metals
Ions
Polymerase Chain Reaction
DNA
Copper
Calcium
Nickel
Aluminum
Iron
Fluorescence Spectrometry
Real-Time Polymerase Chain Reaction
Coloring Agents
Bone and Bones

Keywords

  • 6-Carboxyfluorescein
  • DNA quantification
  • Fluorescence spectroscopy
  • Metal ions
  • PCR inhibition

Cite this

Combs, Laura Gaydosh ; Warren, Joseph E. ; Huynh, Vivian ; Castaneda, Joanna ; Golden, Teresa D. ; Roby, Rhonda K. / The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit. In: Forensic Science International: Genetics. 2015 ; Vol. 19. pp. 180-189.
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abstract = "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{\circledR} Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler{\circledR} 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{\circledR} 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{\circledR} 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.",
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The effects of metal ion PCR inhibitors on results obtained with the Quantifiler® Human DNA Quantification Kit. / Combs, Laura Gaydosh; Warren, Joseph E.; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D.; Roby, Rhonda K.

In: Forensic Science International: Genetics, Vol. 19, 03.08.2015, p. 180-189.

Research output: Contribution to journalArticleResearchpeer-review

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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.

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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.

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