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具有增强 DNA 扩增能力的工程病毒 DNA 聚合酶：损伤 DNA 等温扩增的概念验证。

Engineered viral DNA polymerase with enhanced DNA amplification capacity: a proof-of-concept of isothermal amplification of damaged DNA.

机构信息

Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, CSIC-UAM, Madrid, Spain.

Department of Biochemistry, Universidad Autónoma de Madrid and Instituto de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, Madrid, Spain.

出版信息

Sci Rep. 2020 Sep 14;10(1):15046. doi: 10.1038/s41598-020-71773-6.

DOI:10.1038/s41598-020-71773-6
PMID:32929102
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7490695/
Abstract

The development of whole genome amplification (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses. This has led to a continual requirement of improved DNA amplification protocols and the elaboration of new tailored methods. As key elements in WGA, identification and engineering of novel, faithful and processive DNA polymerases is a driving force in the field. We have engineered the B-family DNA polymerase of virus Bam35 with a C-terminal fusion of DNA-binding motifs. The new protein, named B35-HhH, shows faithful DNA replication in the presence of magnesium or an optimised combination of magnesium and manganese divalent cofactors, which enhances the replication of damaged DNA substrates. Overall, the newly generated variant displays improved amplification performance, sensitivity, translesion synthesis and resistance to salt, which are of great interest for several applications of isothermal DNA amplification. Further, rolling-circle amplification of abasic site-containing minicircles provides a proof-of-concept for using B35-HhH for processive amplification of damaged DNA samples.

摘要

全基因组扩增（WGA）技术及其相关方法的发展，加上测序能力的显著提高，改变了基因组和遗传分析的模式。这导致了对改进的 DNA 扩增方案的持续需求，并详细阐述了新的定制方法。作为 WGA 的关键要素，鉴定和工程新型、忠实和持续的 DNA 聚合酶是该领域的驱动力。我们通过在病毒 Bam35 的 B 族 DNA 聚合酶的 C 端融合 DNA 结合基序，对其进行了工程改造。这种新的蛋白质被命名为 B35-HhH，在镁或优化的镁和锰二价辅因子组合存在的情况下，能够忠实复制 DNA，从而增强了对受损 DNA 底物的复制。总的来说，新生成的变体显示出改进的扩增性能、灵敏度、跨损伤合成和耐盐性，这对等温 DNA 扩增的几种应用非常有意义。此外，含有碱基缺失的小型环的滚环扩增为使用 B35-HhH 进行连续扩增受损 DNA 样本提供了一个概念验证。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/947ea7f29d95/41598_2020_71773_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/5c63064196fd/41598_2020_71773_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/8f01bb5fd46b/41598_2020_71773_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/47160b24c3e4/41598_2020_71773_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/8a6776ad4466/41598_2020_71773_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/01a02a847300/41598_2020_71773_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/d96e434683c1/41598_2020_71773_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/e2bce7271cba/41598_2020_71773_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/947ea7f29d95/41598_2020_71773_Fig8_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/5c63064196fd/41598_2020_71773_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/8f01bb5fd46b/41598_2020_71773_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/47160b24c3e4/41598_2020_71773_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/8a6776ad4466/41598_2020_71773_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/01a02a847300/41598_2020_71773_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/d96e434683c1/41598_2020_71773_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/e2bce7271cba/41598_2020_71773_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/b18e/7490695/947ea7f29d95/41598_2020_71773_Fig8_HTML.jpg

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