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Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

ABSTRACT: Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3' end of the primer, we observed significant decreases in the analytical sensitivity (0-4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer-template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications.

SUBMITTER: Huang K

PROVIDER: S-EPMC10888005 | biostudies-literature | 2024 Feb

REPOSITORIES: biostudies-literature

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Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Huang Ke K Zhang Jilei J Li Jing J Qiu Haixiang H Wei Lanjing L Yang Yi Y Wang Chengming C

Genes 20240207 2

Polymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer-template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer-template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different ...[more]

PMID: 38397205

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Project description:The polymerase chain reaction (PCR) is sensitive to mismatches between primer and template, and mismatches can lead to inefficient amplification of targeted regions of DNA template. In PCRs in which a degenerate primer pool is employed, each primer can behave differently. Therefore, inefficiencies due to different primer melting temperatures within a degenerate primer pool, in addition to mismatches between primer binding sites and primers, can lead to a distortion of the true relative abundance of targets in the original DNA pool. A theoretical analysis indicated that a combination of primer-template and primer-amplicon interactions during PCR cycles 3-12 is potentially responsible for this distortion. To test this hypothesis, we developed a novel amplification strategy, entitled "Polymerase-exonuclease (PEX) PCR", in which primer-template interactions and primer-amplicon interactions are separated. The PEX PCR method substantially and significantly improved the evenness of recovery of sequences from a mock community of known composition, and allowed for amplification of templates with introduced mismatches near the 3' end of the primer annealing sites. When the PEX PCR method was applied to genomic DNA extracted from complex environmental samples, a significant shift in the observed microbial community was detected. Furthermore, the PEX PCR method provides a mechanism to identify which primers in a primer pool are annealing to target gDNA. Primer utilization patterns revealed that at high annealing temperatures in the PEX PCR method, perfect match annealing predominates, while at lower annealing temperatures, primers with up to four mismatches with templates can contribute substantially to amplification. The PEX PCR method is simple to perform, is limited to PCR mixes and a single exonuclease step which can be performed without reaction cleanup, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

Dataset Information

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Publications

Exploring the Impact of Primer-Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity.

Similar Datasets

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