Challenging the proposed causes of the PCR plateau phase

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Jansson, LindaLund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH,Swedish National Forensic Center (författare)

Challenging the proposed causes of the PCR plateau phase

Artikel/kapitelEngelska2019

Förlag, utgivningsår, omfång ...

Elsevier BV,2019

Nummerbeteckningar

LIBRIS-ID:oai:lup.lub.lu.se:2f14abec-1299-495b-bc01-93c772040442
https://lup.lub.lu.se/record/2f14abec-1299-495b-bc01-93c772040442URI
https://doi.org/10.1016/j.bdq.2019.100082DOI

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Språk:engelska
Sammanfattning på:engelska

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Ämneskategori:art swepub-publicationtype
Ämneskategori:ref swepub-contenttype

Anmärkningar

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.

Ämnesord och genrebeteckningar

NATURVETENSKAP Biologi Biokemi och molekylärbiologi hsv//swe
NATURAL SCIENCES Biological Sciences Biochemistry and Molecular Biology hsv//eng
Amplicon yield
Amplification efficiency
DNA polymerase
PCR
Plateau phase
qPCR

Biuppslag (personer, institutioner, konferenser, titlar ...)

Hedman, JohannesLund University,Lunds universitet,Teknisk mikrobiologi,Centrum för tillämpade biovetenskaper,Kemiska institutionen,Institutioner vid LTH,Lunds Tekniska Högskola,Applied Microbiology,Center for Applied Life Sciences,Department of Chemistry,Departments at LTH,Faculty of Engineering, LTH,Swedish National Forensic Center(Swepub:lu)tmb-jeh (författare)
Teknisk mikrobiologiCentrum för tillämpade biovetenskaper (creator_code:org_t)

Sammanhörande titlar

Ingår i:Biomolecular Detection and Quantification: Elsevier BV172214-7535

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Biomolecular Detection and Quantification (Sök värdpublikationen i LIBRIS)

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Av författaren/redakt...: Jansson, Linda; Hedman, Johannes

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NATURVETENSKAP: NATURVETENSKAP; och Biologi; och Biokemi och mole ...

Artiklar i publikationen: Biomolecular Det ...

Av lärosätet: Lunds universitet

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