Schematic representation of a cloning strategy using a Body Double ENase when the Type IIP recognition sites in the vector are not reconstituted by the ligation at <i>A.)</i> one of the ends or <i>B.)</i> both ends of the insert.

<p>Digestion with a Body Double Type IIS restriction endonuclease creates the appropriate overhangs on the PCR fragment that are identical to the overhangs generated by the Type IIP ENase. <b>A.</b>) Only one of the REP restriction sites is reconstituted by the ligation at one end of the insert, and thus a unique REP site is formed in the vector. <b>B.</b>) The REP restriction sites are not reconstituted by the ligation at either ends of the insert. No REP site remains in the vector. REP: Type IIP restriction enzyme, its recognition site is represented by orange boxes. The sequences between the cutting positions corresponding to the overhangs are indicated by altered coloring in the boxes. RES: Body Double Type IIS restriction endonuclease, its recognition site is represented by green boxes. The flanking bases needed for efficient enzymatic digestion of the PCR fragment are marked in light grey.