Error-suppression mechanism of PCR by blocker strands.
Journal
Biophysical journal
ISSN: 1542-0086
Titre abrégé: Biophys J
Pays: United States
ID NLM: 0370626
Informations de publication
Date de publication:
04 04 2023
04 04 2023
Historique:
received:
06
11
2022
revised:
22
01
2023
accepted:
19
02
2023
pmc-release:
04
04
2024
medline:
7
4
2023
pubmed:
25
2
2023
entrez:
24
2
2023
Statut:
ppublish
Résumé
The polymerase chain reaction (PCR) is a central technique in biotechnology. Its ability to amplify a specific target region of a DNA sequence has led to prominent applications, including virus tests, DNA sequencing, genotyping, and genome cloning. These applications rely on the specificity of the primer hybridization and therefore require effective suppression of hybridization errors. A simple and effective method to achieve that is to add blocker strands, also called clamps, to the PCR mixture. These strands bind to the unwanted target sequence, thereby blocking the primer mishybridization. Because of its simplicity, this method is applicable to a broad nucleic-acid-based biotechnology. However, the precise mechanism by which blocker strands suppress PCR errors remains to be understood, limiting the applicability of this technique. Here, we combine experiments and theoretical modeling to reveal this mechanism. We find that the blocker strands both energetically destabilize the mishybridized complex and sculpt a kinetic barrier to suppress mishybridization. This combination of energetic and kinetic biasing extends the viable range of annealing temperatures, which reduces design constraint of the primer sequence and extends the applicability of PCR.
Identifiants
pubmed: 36823986
pii: S0006-3495(23)00134-0
doi: 10.1016/j.bpj.2023.02.028
pmc: PMC10111364
pii:
doi:
Substances chimiques
DNA Primers
0
Nucleic Acids
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1334-1341Informations de copyright
Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of interests The authors declare no competing interests.
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