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PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions

Sidstedt, Maja LU ; Rådström, Peter LU and Hedman, Johannes LU (2020) In Analytical and Bioanalytical Chemistry
Abstract

DNA analysis has seen an incredible development in terms of instrumentation, assays and applications over the last years. Massively parallel sequencing (MPS) and digital PCR are now broadly applied in research and diagnostics, and quantitative PCR is used for more and more practises. All these techniques are based on in vitro DNA polymerization and fluorescence measurements. A major limitation for successful analysis is the various sample-related substances that interfere with the analysis, i.e. PCR inhibitors. PCR inhibition affects library preparation in MPS analysis and skews quantification in qPCR, and some inhibitors have been found to quench the fluorescence of the applied fluorophores. Here, we provide a deeper understanding of... (More)

DNA analysis has seen an incredible development in terms of instrumentation, assays and applications over the last years. Massively parallel sequencing (MPS) and digital PCR are now broadly applied in research and diagnostics, and quantitative PCR is used for more and more practises. All these techniques are based on in vitro DNA polymerization and fluorescence measurements. A major limitation for successful analysis is the various sample-related substances that interfere with the analysis, i.e. PCR inhibitors. PCR inhibition affects library preparation in MPS analysis and skews quantification in qPCR, and some inhibitors have been found to quench the fluorescence of the applied fluorophores. Here, we provide a deeper understanding of mechanisms of specific PCR inhibitors and how these impact specific analytical techniques. This background knowledge is necessary in order to take full advantage of modern DNA analysis techniques, specifically for analysis of samples with low amounts of template and high amounts of background material. The classical solution to handle PCR inhibition is to purify or dilute DNA extracts, which leads to DNA loss. Applying inhibitor-tolerant DNA polymerases, either single enzymes or blends, provides a more straightforward and powerful solution. This review includes mechanisms of specific PCR inhibitors as well as solutions to the inhibition problem in relation to cutting-edge DNA analysis.

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publication status
published
subject
keywords
Blood, Digital PCR, DNA polymerase, Humic substances, Massively parallel sequencing, PCR inhibitors
in
Analytical and Bioanalytical Chemistry
publisher
Springer
external identifiers
  • scopus:85079419696
  • pmid:32052066
ISSN
1618-2642
DOI
10.1007/s00216-020-02490-2
language
English
LU publication?
yes
id
d298840f-13da-411c-9b77-0d03d661f8cb
date added to LUP
2020-02-28 12:42:05
date last changed
2021-06-02 01:28:24
@article{d298840f-13da-411c-9b77-0d03d661f8cb,
  abstract     = {<p>DNA analysis has seen an incredible development in terms of instrumentation, assays and applications over the last years. Massively parallel sequencing (MPS) and digital PCR are now broadly applied in research and diagnostics, and quantitative PCR is used for more and more practises. All these techniques are based on in vitro DNA polymerization and fluorescence measurements. A major limitation for successful analysis is the various sample-related substances that interfere with the analysis, i.e. PCR inhibitors. PCR inhibition affects library preparation in MPS analysis and skews quantification in qPCR, and some inhibitors have been found to quench the fluorescence of the applied fluorophores. Here, we provide a deeper understanding of mechanisms of specific PCR inhibitors and how these impact specific analytical techniques. This background knowledge is necessary in order to take full advantage of modern DNA analysis techniques, specifically for analysis of samples with low amounts of template and high amounts of background material. The classical solution to handle PCR inhibition is to purify or dilute DNA extracts, which leads to DNA loss. Applying inhibitor-tolerant DNA polymerases, either single enzymes or blends, provides a more straightforward and powerful solution. This review includes mechanisms of specific PCR inhibitors as well as solutions to the inhibition problem in relation to cutting-edge DNA analysis.</p>},
  author       = {Sidstedt, Maja and Rådström, Peter and Hedman, Johannes},
  issn         = {1618-2642},
  language     = {eng},
  month        = {02},
  publisher    = {Springer},
  series       = {Analytical and Bioanalytical Chemistry},
  title        = {PCR inhibition in qPCR, dPCR and MPS—mechanisms and solutions},
  url          = {http://dx.doi.org/10.1007/s00216-020-02490-2},
  doi          = {10.1007/s00216-020-02490-2},
  year         = {2020},
}