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

[Mary Wilson]

​Virologica Sinica

Available online 28 February 2022

https://doi.org/10.1016/j.virs.2022.02.009

LeiYang[SUP]a[/SUP][SUP]b[/SUP]LingqianTian[SUP]a[/SUP][SUP]b[/SUP]LeshanLi[SUP]a[/SUP][SUP]b[/SUP]QiuhongLiu[SUP]a[/SUP][SUP]b[/SUP]XiangGuo[SUP]a[/SUP][SUP]b[/SUP]YuanZhou[SUP]a[/SUP]RongjuanPei[SUP]a[/SUP]XinwenChen[SUP]a[/SUP]YunWang[SUP]a[/SUP]

Highlights

•The first TAR assembly system using dual-selection markers.
•Highly efficient TAR assembly system with a zero rate of vector recircularization.
•Assembly of an MPXV genomic fragment containing multiple qPCR detection targets.

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 P[SUB]ADH1[/SUB]-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 cerevisiaestrain, VL6-48B, was prepared by chromosomal substitution of ura3-52with 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.

https://www.sciencedirect.com/science/article/pii/S1995820X22000414#!