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Challenging the proposed causes of the PCR plateau phase

Jansson, Linda LU and Hedman, Johannes LU (2019) In Biomolecular Detection and Quantification 17.
Abstract

Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. By applying robust and validated qPCR assays, we elucidate the impact of adding non-target and target... (More)

Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. By applying robust and validated qPCR assays, we elucidate the impact of adding non-target and target amplicons to the reactions, mimicking the high amount of products in late PCR cycles. Further, the impact of increased primer concentrations and thermal stability of reagents are explored. Our results show that high amounts of non-target amplicons inhibit amplification by binding to the DNA polymerase, but that this effect is counteracted by addition of more DNA polymerase or prolonged annealing/extension times. Adding high amounts of target amplicons that also act as templates in the reaction is far less inhibitory to amplification, although a decrease in amplification rate is seen. When primer concentrations are increased, both amplification rates and end-product yields are elevated. Taken together, our results suggest that the main cause of PCR plateau formation is primer depletion and not product accumulation or degradation of reagents. We stress that a PCR plateau caused by primer depletion is assay-dependent, i.e. dependent on the primer design and primer characteristics such as the probability of primer-dimer formation. Our findings contribute to an improved understanding of the major parameters controlling the PCR dynamics at later cycles and the limitations of continued product formation, which in the end can facilitate PCR optimization.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Amplicon yield, Amplification efficiency, DNA polymerase, PCR, Plateau phase, qPCR
in
Biomolecular Detection and Quantification
volume
17
publisher
Elsevier GmbH
external identifiers
  • scopus:85062239203
ISSN
2214-7535
DOI
10.1016/j.bdq.2019.100082
language
English
LU publication?
yes
id
2f14abec-1299-495b-bc01-93c772040442
date added to LUP
2019-03-11 11:07:06
date last changed
2019-04-02 04:14:42
@article{2f14abec-1299-495b-bc01-93c772040442,
  abstract     = {<p>Despite the wide-spread use of the polymerase chain reaction (PCR) in various life-science applications, the causes of arrested amplicon generation in late cycles have not been confidently identified. This so-called plateau phase has been attributed to depletion or thermal break-down of primers or nucleotides, thermal inactivation of the DNA polymerase, and product accumulation resulting in competition between primer annealing and product re-hybridization as well as blocking of DNA polymerase by double-stranded amplicons. In the current study, we experimentally investigate the proposed limiting factors of PCR product formation. By applying robust and validated qPCR assays, we elucidate the impact of adding non-target and target amplicons to the reactions, mimicking the high amount of products in late PCR cycles. Further, the impact of increased primer concentrations and thermal stability of reagents are explored. Our results show that high amounts of non-target amplicons inhibit amplification by binding to the DNA polymerase, but that this effect is counteracted by addition of more DNA polymerase or prolonged annealing/extension times. Adding high amounts of target amplicons that also act as templates in the reaction is far less inhibitory to amplification, although a decrease in amplification rate is seen. When primer concentrations are increased, both amplification rates and end-product yields are elevated. Taken together, our results suggest that the main cause of PCR plateau formation is primer depletion and not product accumulation or degradation of reagents. We stress that a PCR plateau caused by primer depletion is assay-dependent, i.e. dependent on the primer design and primer characteristics such as the probability of primer-dimer formation. Our findings contribute to an improved understanding of the major parameters controlling the PCR dynamics at later cycles and the limitations of continued product formation, which in the end can facilitate PCR optimization.</p>},
  articleno    = {100082},
  author       = {Jansson, Linda and Hedman, Johannes},
  issn         = {2214-7535},
  keyword      = {Amplicon yield,Amplification efficiency,DNA polymerase,PCR,Plateau phase,qPCR},
  language     = {eng},
  publisher    = {Elsevier GmbH},
  series       = {Biomolecular Detection and Quantification},
  title        = {Challenging the proposed causes of the PCR plateau phase},
  url          = {http://dx.doi.org/10.1016/j.bdq.2019.100082},
  volume       = {17},
  year         = {2019},
}