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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

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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

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Thank You, Other Wise Sister

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“O come, let us worship and bow down: let us kneel before the LORD our maker.”  Psalms 95:6 (KJV) 

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Listen to the article below

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[fusion_dropcap boxed=”no” boxed_radius=”” class=”” id=”” color=”” text_color=””]A[/fusion_dropcap]bstract

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.

Keywords

Monkeypox virusTransformation-associated recombination (TAR)TAR assembly

1. Introduction

One of the characteristic features of yeast is that exogenous DNA fragments can be efficiently taken up and recombined. Typically, two linearized DNA fragments with 60 base pairs (bp) of overlapping sequences can be readily recombined and ligated by homologous recombination (HR) in yeast (Noskov et al., 2001). Based on this feature, transformation-associated recombination (TAR) was developed. TAR has shown great value in the isolation of chromosomal fragments from the genomic DNA pool (TAR cloning), as well as in the assembly of multiple DNA fragments (TAR assembly) into a single yeast or bacterial artificial chromosome (YAC or BAC) [reviewed in (Kouprina and Larionov 2016)]. The basic approach of TAR is to use a linearized vector to capture DNA of interest by “hook” sequences through HR after they have been cotransformed into yeast cells. However, the efficiency of TAR can be severely hampered by error-prone DNA repair pathways, including but not limited to the nonhomologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) [reviewed in (Lewis and Resnick, 2000)]. It was estimated that at least 10%–80% of yeast transformants contain false TAR products, and a considerable fraction is attributed to vector recircularization (Kuijpers et al., 2013).

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