Practice question

YAC cloning involves ligation of insert DNA between two arms each carrying telomere, selection marker and centromere. Linearized vector without insert can recircularize through non-homologous end joining, regenerating empty YAC. SUP4, encoding suppressor tRNA for ochre mutation, serves as distinguishing marker. When SUP4 is interrupted by insert, suppressor phenotype is lost, producing red colonies in ade2-101 background, while religated empty vector retains functional SUP4 giving white colonies. CEN, ARS, URA3, TRP1 present in both forms cannot differentiate. SUP4 inactivation provides positive visual indication of recombinant formation versus vector background.