Fill-In Reaction

 DNA fragments with 5′-overhanging ends are converted to blunt-ended molecules by reacting with the Klenow fragment of Escherichia coli DNA polymerase. This makes the overhanging ends double-stranded and is called the fill-in reaction. The fill-in reaction is often used to join fragments with different cohesive ends by converting the cohesive ends to blunt ends. This reaction is also used to introduce a frameshift mutation into a gene. A plasmid containing the gene is cut within the gene using a restriction enzyme to produce ends with a 5′-overhang. The overhang is filled in and the ends are religated. As long as the length of the overhang is not divisible by three, this procedure changes the reading frame of the gene downstream of the restriction site.