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- Sidstedt, Maja, et al.
(författare)
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Digital PCR inhibition mechanisms using standardized inhibitors representing soil and blood matrices
- 2016
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentrationis determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrices such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. Here, we present how certain inhibitors disturb dPCR quantification and suggest solutions to these problems. Furthermore, we use real-time PCR, dPCR and isothermal titration calorimetry as tools to elucidate the mechanisms underlying the PCR inhibition. The impact of impurities on dPCR quantification was studied using humic acid as a model inhibitor. We show that the inhibitor-tolerance differs greatly for three different DNA polymerases, illustrating the importance of choosing a DNA polymerase-buffer system that is compatible with the samples to be analysed. Various inhibitory-substances from blood were found to disturb the system in different ways. For example, hemoglobin was found to cause quenching of fluorescence and a dramatic decrease of the number of positive reactions, leading to an underestimation of DNA quantity. IgG caused an increased number of late-starters. The system was more susceptible to inhibition by IgG when single-stranded DNA was used as template, compared with double-stranded DNA. By understanding more about the mechanisms of PCR inhibitors it will be possible to design more optimal PCR chemistries, improving dPCR detection and quantification.
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| 2. |
- Sidstedt, Maja, et al.
(författare)
-
Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR
- 2018
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Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 410:10, s. 2569-2583
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Tidskriftsartikel (refereegranskat)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 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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