Indian Journal of Experimental Biology Vol. 57, December 2019, pp. 945-948 KasI vs. SspDI: Comparative analysis of enzyme activity for restriction digestion Suman, Rekha Puria & Vikrant Nain* School of Biotechnology, Gautam Buddha University, Greater Noida-201 312, Uttar Pradesh, India Received 09 March 2017; revised 08 August 2019 Type II restriction enzymes are routinely used by molecular biologists in designing and implementation of cloning experiments without referring to the literature on enzymes in use, and at times, face some unforeseeable problems. In our laboratory too, we encountered one such problem while working with KasI restriction enzyme which recognizes GGCGCC sequence, and we further analyzed the issue. Our observations corroborate the fact that KasI acts as monomer and cleaves double stranded DNA through nicking mechanism. It introduces breaks in two strands of DNA after substantial time gap which can be owed to two independent nickase activities in the opposite strands. Moreover, this time gap between two nickase activities results in formation of different topological forms of DNA. Since molecular biologists working with common restriction enzymes are not familiar with such nickase activity, they may misinterpret their restriction digestion results. However, no such problem was observed with the use of SspDI restriction enzyme which also recognizes the same sequence (GGCGCC) and produces the identical overhangs as by KasI. Hence, SspDI suits better for routine cloning and genetic modification purposes over KasI while using GGCGCC as cloning site. Keywords: Cloning, DNA modifying enzyme, Recombinant DNA, Restriction enzyme The discovery of restriction endonucleases, that are naturally a part of bacterial defence mechanism against invading nucleic acids (viruses) 1 , has ushered in a new era of recombinant DNA technology 2-6 . These restriction enzymes, particularly type II restriction enzymes that have well defined recognition and cleavage sites, have become indispensable for any molecular biology laboratory. The quest for genetic modification for basic as well as applied research has led to the identification of restriction enzymes with different recognition sequences. More than 4500 restriction enzymes are known today and this number is ever increasing 7 . Interestingly, these thousands of restriction enzymes do not show sequence similarity among them, and hence are believed to have evolved independently 8-11 . Due to this independent evolution of their protein sequences, restriction enzymes have adopted different mechanisms of sequence recognition and cleavage e.g., difference in cleavage site, methylation state, influence of flanking sequence, requirement of cofactor and specific salt and in some cases presence of two recognition sites (cis or trans) 8,10-15 . Although, factors affecting the activity of a particular restriction enzyme are studied in detail for their optimization before commercialization and releasing them for use in the laboratories, still certain unpredictable technical issues may arise only when these are put in application in laboratories. In our laboratory, we encountered such problem with KasI restriction enzyme when used for cleaving at GGCGCC site. Hence, here, we studied the activity and efficacy of the KasI and SspDI restriction enzymes for digestion at GGCGCC site. Materials and Methods Plasmid vectors A 207 bp DNA fragment was cloned in pUC57 vector using EcoRI and HindIII sites. KasI and SmaI restriction sites were included in the cloned sequence downstream to EcoRI site and upstream to HindIII site, respectively. The fragment of 195 bp having KasI and SmaI as flanking sites was further required to be cloned in another vector using KasI at 5' end and SmaI at 3' end. Eight restriction digestion reactions each for digestion of 50 ng plasmid DNA in a 20 μL reaction volume using 2.5 units of KasI enzyme were set up and incubated for different time intervals (from 10 min to 48 h) as mentioned in the experiment. —————— *Correspondence: Phone: +91 120 2344283; Fax: +91 120 2344205 E-Mail: vikrant@gbu.ac.in