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. doi: 10.1016/j.virs.2022.02.009
Citation: Lei Yang, Lingqian Tian, Leshan Li, Qiuhong Liu, Xiang Guo, Yuan Zhou, Rongjuan Pei, Xinwen Chen, Yun Wang. Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination [J].VIROLOGICA SINICA, 2022, 37(3) : 341-347.  http://dx.doi.org/10.1016/j.virs.2022.02.009

双向筛选TAR系统可以有效组装用于qPCR检测的MPXV基因组片段模板

  • 转化相关重组(TAR)已被广泛用于组装较大DNA产物,而影响组装效率的障碍之一是酵母中存在错误修复DNA途径,这导致载体骨架自连或非正确重组产物的产生。为了提高TAR组装效率,我们制备了一种双向筛选载体PGFCS,通过将PADH1-URA3添加到细菌酵母人工染色体PGF,含有PHIS3-HIS3盒作为阳性选择标记,以添加的PADH1-URA3用作负选择标记,以确保携带线性化载体自连的酵母不能在5-氟乳清酸(5-FOA)存在下生长。为了防止PGFCS中引入的ura3与酵母基因组中的内源ura3-52重组,利用Blasticidin的抗生素抗性基因替代酵母染色体中的ura3-52,制备高度可转化的酿酒酵母VL6-48B。在VL6-48B中利用PGFCS,通过TAR组装了包含定量PCR的主要检测靶标-猴痘病毒的55kb基因组片段。PGFCS介导的TAR重组显示载体循环化率为零,正确组装产率均值为79%,表明双选策略可优化TAR重组。

Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

  • Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a PHIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

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    Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

      Corresponding author: Xinwen Chen, chenxw@wh.iov.cn
      Corresponding author: Yun Wang, wangyun@wh.iov.cn
    • a State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, 430071, China;

    Abstract: Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a PADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a PHIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.

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