生物
聚合酶
DNA聚合酶
基因组
衣壳
基因
腺相关病毒
多重位移放大
计算生物学
遗传学
聚合酶链反应
DNA
病毒学
载体(分子生物学)
重组DNA
DNA提取
作者
Qiang Wang,Kalyani Nambiar,James M. Wilson
出处
期刊:Human Gene Therapy
[Mary Ann Liebert]
日期:2021-12-01
卷期号:32 (23-24): 1439-1449
被引量:3
摘要
Adeno-associated viruses (AAVs) are advantageous as gene-transfer vectors due to their favorable biological and safety characteristics, with discovering novel AAV variants being key to improving this treatment platform. To date, researchers have isolated over 200 AAVs from natural sources using PCR-based methods. We compared two modern DNA polymerases and their utility for isolating and amplifying the AAV genome. Compared to the HotStar polymerase, the higher-fidelity Q5 Hot Start High-Fidelity DNA Polymerase provided more precise and accurate amplification of the input AAV sequences. The lower-fidelity HotStar DNA polymerase introduced mutations during the isolation and amplification processes, thus generating multiple mutant capsids with variable bioactivity compared to the input AAV gene. The Q5 polymerase enabled the successful discovery of novel AAV capsid sequences from human and nonhuman primate tissue sources. Novel AAV sequences from these sources showed evidence of positive evolutionary selection. This study highlights the importance of using the highest fidelity DNA polymerases available to accurately isolate and characterize AAV genomes from natural sources to ultimately develop more effective gene therapy vectors.
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