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Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase
Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improv...
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/PMC10347390/ https://www.ncbi.nlm.nih.gov/pubmed/37456715 http://dx.doi.org/10.3389/fbioe.2023.1233856 |
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author | Zhang, Jia Su, Xiaolu Wang, Yefei Wang, Xiaohang Zhou, Shiqi Jia, Hui Jing, Xiaoyan Gong, Yanhai Wang, Jichao Xu, Jian |
author_facet | Zhang, Jia Su, Xiaolu Wang, Yefei Wang, Xiaohang Zhou, Shiqi Jia, Hui Jing, Xiaoyan Gong, Yanhai Wang, Jichao Xu, Jian |
author_sort | Zhang, Jia |
collection | PubMed |
description | Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing. |
format | Online Article Text |
id | pubmed-10347390 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-103473902023-07-15 Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase Zhang, Jia Su, Xiaolu Wang, Yefei Wang, Xiaohang Zhou, Shiqi Jia, Hui Jing, Xiaoyan Gong, Yanhai Wang, Jichao Xu, Jian Front Bioeng Biotechnol Bioengineering and Biotechnology Single-cell genomic whole genome amplification (WGA) is a crucial step in single-cell sequencing, yet its low amplification efficiency, incomplete and uneven genome amplification still hinder the throughput and efficiency of single-cell sequencing workflows. Here we introduce a process called Improved Single-cell Genome Amplification (iSGA), in which the whole single-cell sequencing cycle is completed in a high-efficient and high-coverage manner, through phi29 DNA polymerase engineering and process engineering. By establishing a disulfide bond of F137C-A377C, the amplification ability of the enzyme was improved to that of single-cell. By further protein engineering and process engineering, a supreme enzyme named HotJa Phi29 DNA Polymerase was developed and showed significantly better coverage (99.75%) at a higher temperature (40°C). High single-cell genome amplification ability and high coverage (93.59%) were also achieved for commercial probiotic samples. iSGA is more efficient and robust than the wild-type phi29 DNA polymerase, and it is 2.03-fold more efficient and 10.89-fold cheaper than the commercial Thermo Scientific EquiPhi29 DNA Polymerase. These advantages promise its broad applications in large-scale single-cell sequencing. Frontiers Media S.A. 2023-06-29 /pmc/articles/PMC10347390/ /pubmed/37456715 http://dx.doi.org/10.3389/fbioe.2023.1233856 Text en Copyright © 2023 Zhang, Su, Wang, Wang, Zhou, Jia, Jing, Gong, Wang and Xu. 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 Zhang, Jia Su, Xiaolu Wang, Yefei Wang, Xiaohang Zhou, Shiqi Jia, Hui Jing, Xiaoyan Gong, Yanhai Wang, Jichao Xu, Jian Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title | Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title_full | Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title_fullStr | Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title_full_unstemmed | Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title_short | Improved single-cell genome amplification by a high-efficiency phi29 DNA polymerase |
title_sort | improved single-cell genome amplification by a high-efficiency phi29 dna polymerase |
topic | Bioengineering and Biotechnology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10347390/ https://www.ncbi.nlm.nih.gov/pubmed/37456715 http://dx.doi.org/10.3389/fbioe.2023.1233856 |
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