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Title: KasI vs. SspDI: Comparative analysis of enzyme activity for restriction digestion
Authors: Suman
Puria, Rekha
Nain, Vikrant
Keywords: Cloning;DNA modifying enzyme;Recombinant DNA;Restriction enzyme
Issue Date: Dec-2019
Publisher: NISCAIR-CSIR, India
Abstract: 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.
Page(s): 945-948
ISSN: 0975-1009 (Online); 0019-5189 (Print)
Appears in Collections:IJEB Vol.57(12) [December 2019]

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