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...
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1180542/full |
| _version_ | 1797838557473144832 |
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| author | Yongsong Zhou Yongsong Zhou Fan Bo Tian Tian Buling Wu Bofeng Zhu Bofeng Zhu Bofeng Zhu |
| author_facet | Yongsong Zhou Yongsong Zhou Fan Bo Tian Tian Buling Wu Bofeng Zhu Bofeng Zhu Bofeng Zhu |
| author_sort | Yongsong Zhou |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-04-09T15:42:45Z |
| format | Article |
| id | doaj.art-220cfa75b71b4e5f9dccfd3643865792 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-04-09T15:42:45Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-220cfa75b71b4e5f9dccfd36438657922023-04-27T05:33:51ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-04-011110.3389/fbioe.2023.11805421180542Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplificationYongsong Zhou0Yongsong Zhou1Fan Bo2Tian Tian3Buling Wu4Bofeng Zhu5Bofeng Zhu6Bofeng Zhu7Shenzhen Stomatology Hospital (Pingshan), Southern Medical University, Shenzhen, Guangdong, ChinaGuangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou, Guangdong, ChinaGuangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou, Guangdong, ChinaShenzhen Stomatology Hospital (Pingshan), Southern Medical University, Shenzhen, Guangdong, ChinaShenzhen Stomatology Hospital (Pingshan), Southern Medical University, Shenzhen, Guangdong, ChinaShenzhen Stomatology Hospital (Pingshan), Southern Medical University, Shenzhen, Guangdong, ChinaGuangzhou Key Laboratory of Forensic Multi-Omics for Precision Identification, School of Forensic Medicine, Southern Medical University, Guangzhou, Guangdong, ChinaKey Laboratory of Shaanxi Province for Craniofacial Precision Medicine Research, College of Stomatology, Xi’an Jiaotong University, Xi’an, Shanxi, ChinaBecause 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.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1180542/fullnon-template additionTaq DNA polymeraseartifactsplit peakcapillary electrophoresis |
| spellingShingle | Yongsong Zhou Yongsong Zhou Fan Bo Tian Tian Buling Wu Bofeng Zhu Bofeng Zhu Bofeng Zhu Excessive addition split peak formed by the non-templated nucleotide addition property of Taq DNA polymerase after PCR amplification Frontiers in Bioengineering and Biotechnology non-template addition Taq DNA polymerase artifact split peak capillary electrophoresis |
| 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 | non-template addition Taq DNA polymerase artifact split peak capillary electrophoresis |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1180542/full |
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