Advances in Biochemistry and Biotechnology (ISSN: 2574-7258)

Review Article

ATM: Adenine, Thymine Mediated Ligase Free Ligation

Rajagopal Kammara1*, Lakshmanan Vinaytheerthan2

1AcSIR, and Department of Protein Chemistry and Technology (PCT), Central Food Technological Research Institute (CSIR-CFTRI), Mysore, India

2LV a Junior Research Fellow under DST-SERB project, India

Corresponding Author*: Rajagopal Kammara, AcSIR and Department of Protein Chemistry and Technology (PCT), Central Food Technological Research Institute (CSIR-CFTRI), Mysore, India.

Received Date: 06 September, 2021

Accepted Date: 21 September, 2021

Published Date: 24 September, 2021

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.


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