Creating the insert
The insert is created by PCR using Taq DNA polymerase. This polymerase lacks 3' to 5' proofreading activity and, with a high probability, adds a single, 3'-adenine overhang to each end of the PCR product. It is best if the PCR primers have guanines at the 5' end as this maximizes probability of Taq DNA polymerase adding the terminal adenosine overhang.[2] Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity should not be used as they do not leave the 3´ adenine-overhangs.