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

Maja Sidstedt, Johannes Hedman, Erica L. Romsos, Leticia Waitara, Lars Wadsö, Carolyn R. Steffen, Peter M. Vallone, Peter Rådström

Research output: Contribution to journalArticlepeer-review


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.]

Original languageEnglish
Pages (from-to)2569-2583
JournalAnalytical and Bioanalytical Chemistry
Issue number10
Early online date2018 Mar 5
Publication statusPublished - 2018

Subject classification (UKÄ)

  • Hematology
  • Medicinal Chemistry

Free keywords

  • Blood
  • Digital PCR
  • DNA polymerase
  • PCR inhibition
  • PCR inhibitors
  • Real-time PCR


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