Reparation of DNA sticky ends

The aim of this procedure is to have blunt ends in the target DNA. After the fragmentation step, target DNA has sticky ends. This means that one strand is longer than the other, resulting in either a 3' or 5' overhang. This is due to the sonication process since the way it shears the DNA is not as specific as using restriction enzymes.

Before repairing the sticky ends, the sample should be treated with a special RNase H, in order to degrade any remaining RNA that could compete later with the target DNA during the sequencing step.

Two enzymes will repair the sticky ends:

  • T4 DNA polymerase, which cuts DNA fragments containing both 3' and 5' overhangs.

  • T4 Polynucleotide kinase, which phosphorylates 5' ends.

The result of this treatment is DNA with blunt-ends.