Schematic diagram of IRDL cloning.

<p>A, Construct expression of one insert by IRDL cloning. The purified PCR products of GOI and expression vector are mixed in a single tube together with restriction enzymes 1 and 2, as well as ligase. When the digestion and ligation of the mixture are in balance, two possible ligation products appear: (1) and (2). Only <i>E. coli</i> harbored the desired product (2) by its ability to survive on selection plates after transformation. Since the two different restriction enzymes used will generate two incompatible DNA ends, only the appropriate inserts can be ligated with the expression vector. B, Construction of fusion protein by IRDL cloning. The purified products of GOI1, GOI2, and expression vector are mixed in a single tube together with restriction enzymes 1, 2, and 3, as well as ligase. C, Construction of fusion protein by overlap extension PCR and IRDL cloning. GOI, gene fragment of interest. Lethal, lethal gene.