Restriction endonucleases recognize and cleave dsDNA at highly specific nucleotide sequences, or restriction sites. Many restriction enzymes produce short, staggered ends (with 5′ or 3′ overhangs) that can be rejoined by a DNA ligase to regenerate the original recognition sequence (Figure 1). They are referred to as cohesive ends because of the hydrogen bond stabilization of the DNA bases that loosely holds the DNA ends together prior to ligation. The specificity of restriction enzymes enables directional cloning, and the hydrogen bonding of cohesive ends increases the efficiency of cohesive-end ligation by as much as 100X over blunt-end ligation [1].These features make cohesive-end cloning a highly useful method for molecular biology.