ATM: Adenine, Thymine Mediated Ligase Free Ligation
Advances in Biochemistry and Biotechnology (ISSN: 2574-7258), 6 (1):
3 (September 2021)DOI: https://doi.org/10.29011/2574-7258.010106
Abstract
We distinguish a simple method of efficient sequence dependent and natural concatemerization subsequent to the cloning of PCR products bypassing ligation. Tailed PCR primer pairs were employed to create complementary staggered termini with AT (Adenine, Thymine) bases only on the insert / PCR product. The introduced AT bases either in 5’ or 3’ termini were designed to allow effective intermolecular ligation to internalize the terminal restriction sites in a ligase free manner. The presence of
extra nucleotide sequence in the termini does not restrict the subsequent steps of cloning. This ligase free ligation procedure is extremely efficient, and the subsequent cloning not restrained by the presence of an extra nucleotide in either of the termini. The avoidance of ligase based, chimera formation makes the procedure, rapid, reliable and efficient. The process also bypasses the multiple sequence incorporations at the termini to increase the efficiency of restriction digestion