The red arrows indicate the cleavage sites. The red diamond indicates the axis of two-fold rotational symmetry. The blue and yellow colors represent DNA on each side of the PvuII cleavage site. Blunt-ended DNA fragments can be covalently joined together by T4 DNA ligase and ATP (cohesive ends can also be joined together by E. coli DNA ligase and NAD+). Note: All DNA ligases require a 5'-phosphate and a 3'-hydroxyl at the site of phosphodiester formation. Phosphates can be removed with phosphatase (there are several phosphatases: bacterial alkaline phosphatase, calf intestinal phosphatase, and shrimp phosphatase). Phosphates can be added to 5'-hydroxyls using T4 polynucleotide kinase and ATP. Polynucleotide kinase transfers the gamma (last) phosphate of ATP to the 5'-hydrolyl of nucleic acid (this reaction can be used to label oligonucleotides by transfering radioactive phosphate at the gamma position of ATP to the 5'-hydroxyl of the oligonucleotide).