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Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR

Sidstedt, Maja LU ; Hedman, Johannes LU ; Romsos, Erica L. ; Waitara, Leticia ; Wadsö, Lars LU ; Steffen, Carolyn R. ; Vallone, Peter M. and Rådström, Peter LU (2018) In Analytical and Bioanalytical Chemistry 410(10). p.2569-2583
Abstract

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference... (More)

Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. [Figure not available: see fulltext.]

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Blood, Digital PCR, DNA polymerase, PCR inhibition, PCR inhibitors, Real-time PCR
in
Analytical and Bioanalytical Chemistry
volume
410
issue
10
pages
2569 - 2583
publisher
Springer
external identifiers
  • scopus:85042763803
  • pmid:29504082
ISSN
1618-2642
DOI
10.1007/s00216-018-0931-z
language
English
LU publication?
yes
id
7fec4ba5-bae6-42b6-b72a-8d62a69b1553
date added to LUP
2018-03-15 12:10:40
date last changed
2023-04-08 08:59:19
@article{7fec4ba5-bae6-42b6-b72a-8d62a69b1553,
  abstract     = {{<p>Blood samples are widely used for PCR-based DNA analysis in fields such as diagnosis of infectious diseases, cancer diagnostics, and forensic genetics. In this study, the mechanisms behind blood-induced PCR inhibition were evaluated by use of whole blood as well as known PCR-inhibitory molecules in both digital PCR and real-time PCR. Also, electrophoretic mobility shift assay was applied to investigate interactions between inhibitory proteins and DNA, and isothermal titration calorimetry was used to directly measure effects on DNA polymerase activity. Whole blood caused a decrease in the number of positive digital PCR reactions, lowered amplification efficiency, and caused severe quenching of the fluorescence of the passive reference dye 6-carboxy-X-rhodamine as well as the double-stranded DNA binding dye EvaGreen. Immunoglobulin G was found to bind to single-stranded genomic DNA, leading to increased quantification cycle values. Hemoglobin affected the DNA polymerase activity and thus lowered the amplification efficiency. Hemoglobin and hematin were shown to be the molecules in blood responsible for the fluorescence quenching. In conclusion, hemoglobin and immunoglobulin G are the two major PCR inhibitors in blood, where the first affects amplification through a direct effect on the DNA polymerase activity and quenches the fluorescence of free dye molecules, and the latter binds to single-stranded genomic DNA, hindering DNA polymerization in the first few PCR cycles. [Figure not available: see fulltext.]</p>}},
  author       = {{Sidstedt, Maja and Hedman, Johannes and Romsos, Erica L. and Waitara, Leticia and Wadsö, Lars and Steffen, Carolyn R. and Vallone, Peter M. and Rådström, Peter}},
  issn         = {{1618-2642}},
  keywords     = {{Blood; Digital PCR; DNA polymerase; PCR inhibition; PCR inhibitors; Real-time PCR}},
  language     = {{eng}},
  month        = {{03}},
  number       = {{10}},
  pages        = {{2569--2583}},
  publisher    = {{Springer}},
  series       = {{Analytical and Bioanalytical Chemistry}},
  title        = {{Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR}},
  url          = {{http://dx.doi.org/10.1007/s00216-018-0931-z}},
  doi          = {{10.1007/s00216-018-0931-z}},
  volume       = {{410}},
  year         = {{2018}},
}