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Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification
Because of its non-template addition feature, Taq DNA polymerase can catalyze one or more extra nucleotides onto the 3′ terminus of PCR products. An extra peak is observed at DYS391 locus after the PCR products stored for 4 days at 4°C. To explore the formation mechanism of this artifact, PCR primer...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10174434/ https://www.ncbi.nlm.nih.gov/pubmed/37180044 http://dx.doi.org/10.3389/fbioe.2023.1180542 |
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author | Zhou, Yongsong Bo, Fan Tian, Tian Wu, Buling Zhu, Bofeng |
author_facet | Zhou, Yongsong Bo, Fan Tian, Tian Wu, Buling Zhu, Bofeng |
author_sort | Zhou, Yongsong |
collection | PubMed |
description | Because of its non-template addition feature, Taq DNA polymerase can catalyze one or more extra nucleotides onto the 3′ terminus of PCR products. An extra peak is observed at DYS391 locus after the PCR products stored for 4 days at 4°C. To explore the formation mechanism of this artifact, PCR primers and amplicon sequences of Y-STR loci are analyzed, furthermore, PCR products storage conditions and termination of PCR are discussed. The extra peak is a + 2 addition product, which we call excessive addition split peak (EASP). The most significant difference between EASP and the incomplete addition of adenine product is that the size of EASP is about one base larger than the true allele, and the EASP locates on the right side of the real allelic peak. The EASP cannot be eliminated by increasing loading mixture volume and conducting heat denaturation prior to electrophoresis injection. However, the EASP is not observed when the PCR is terminated with ethylenediaminetetraacetic acid or formamide. These findings suggest that formation of EASP is a result of 3′ end non-template extension by Taq DNA polymerase, rather than being the result of DNA fragment secondary structure produced under a suboptimal electrophoresis condition. In addition, the EASP formation is affected by the primer sequences and the storage conditions of PCR products. |
format | Online Article Text |
id | pubmed-10174434 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-101744342023-05-12 Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification Zhou, Yongsong Bo, Fan Tian, Tian Wu, Buling Zhu, Bofeng Front Bioeng Biotechnol Bioengineering and Biotechnology Because of its non-template addition feature, Taq DNA polymerase can catalyze one or more extra nucleotides onto the 3′ terminus of PCR products. An extra peak is observed at DYS391 locus after the PCR products stored for 4 days at 4°C. To explore the formation mechanism of this artifact, PCR primers and amplicon sequences of Y-STR loci are analyzed, furthermore, PCR products storage conditions and termination of PCR are discussed. The extra peak is a + 2 addition product, which we call excessive addition split peak (EASP). The most significant difference between EASP and the incomplete addition of adenine product is that the size of EASP is about one base larger than the true allele, and the EASP locates on the right side of the real allelic peak. The EASP cannot be eliminated by increasing loading mixture volume and conducting heat denaturation prior to electrophoresis injection. However, the EASP is not observed when the PCR is terminated with ethylenediaminetetraacetic acid or formamide. These findings suggest that formation of EASP is a result of 3′ end non-template extension by Taq DNA polymerase, rather than being the result of DNA fragment secondary structure produced under a suboptimal electrophoresis condition. In addition, the EASP formation is affected by the primer sequences and the storage conditions of PCR products. Frontiers Media S.A. 2023-04-27 /pmc/articles/PMC10174434/ /pubmed/37180044 http://dx.doi.org/10.3389/fbioe.2023.1180542 Text en Copyright © 2023 Zhou, Bo, Tian, Wu and Zhu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Bioengineering and Biotechnology Zhou, Yongsong Bo, Fan Tian, Tian Wu, Buling Zhu, Bofeng Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title | Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title_full | Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title_fullStr | Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title_full_unstemmed | Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title_short | Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification |
title_sort | excessive addition split peak formed by the non-templated nucleotide addition property of taq dna polymerase after pcr amplification |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10174434/ https://www.ncbi.nlm.nih.gov/pubmed/37180044 http://dx.doi.org/10.3389/fbioe.2023.1180542 |
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