Fig. 3.

Assembly of an MPXV fragment by TAR. A Diagram of the DNA fragments for TAR assembly. The TAR vector pGFCS was linearized into two PCR amplicons with the primer pairs 60-ADH1/ARS-HIS3 and HIS3-ARS/55483-URA3. The resulting vector parts and four continuous MPXV segments (yellow rectangle) partially overlapped with each other and formed a circular YAC/BAC. The relative location of each primer is indicated in the diagram. B Validation of assembly products by multiplex PCR. Sixteen yeast colonies from pGFCS-transformants were picked, and DNA contents were extracted as the template for multiplex PCR. Five primer pairs annealed to each TAR recombination site's upstream and downstream regions (diagrammed in A) were used to validate the TAR assembly products. The PCR amplicons were subjected to gel electrophoresis. A legitimate assembly product should generate five amplicons by multiplex PCR, sized 582 bp (primer pair: ADH1/411), 291 bp (primer pair: 14136/14426), 411 bp (primer pair: 26501/26911), 348 bp (primer pair: 40873/41220), and 665 bp (primer pair: 55377/URA3) in length. A recircularized vector should generate a single amplicon 653 bp in length. A typical lane containing five correct amplicons is indicated in the red box. The table at the bottom shows the size of each amplicon and the ratio of legitimate assembly products from three independent TAR assembly experiments (TAR#1, #2, and #3). C Genotyping of the assembly product. Restriction pattern of the assembly product. One of the candidate assembly products was extracted from VL6-48B and transformed to EPI300 for high-efficiency amplification. The resulting DNA was extracted and digested by BamHI. The restriction fragments were separated by gel electrophoresis and were compared to the computer-predicted BamHI restriction pattern (left panel).