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Rapid and reliable method for identification of associated endonuclease cleavage and recognition sites
Author(s) -
Jensen T.Ø.,
Kvist T.,
Mikkelsen M.J.,
Westermann P.
Publication year - 2014
Publication title -
letters in applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.698
H-Index - 110
eISSN - 1472-765X
pISSN - 0266-8254
DOI - 10.1111/lam.12238
Subject(s) - cleavage (geology) , endonuclease , identification (biology) , computational biology , biology , genetics , dna , ecology , paleontology , fracture (geology)
One barrier to cross during genetic engineering is the restriction‐modification system found in many bacteria. In this study, we developed a fast and reliable method for mapping the recognition and cleavage site of the restriction endonucleases. C lostridium pasteurianum, a model organism for the study of nitrogen fixation, has been found to harbour at least two restriction‐modification systems including the restriction endonucleases C pa PI , which is an isoschizomer of M boI and C pa AI . Dam‐methylated DNA was used to isolate the activity of C pa AI . Exposing freshly prepared cell lysate to known nucleotide fragments and directly sequencing the pool of digested nucleotide fragments enabled identification of the cleavage sites in the fragments. By aligning the sequences adjacent to the cleavage site, it was possible to identify the recognition sequence. Using this method, we successfully located all C pa AI recognition and cleavage sites within the template sequence. By modifying DNA with both D am and C p G methylases ( M . S ssI) and thereby preventing digestion by C pa PI and C pa AI , no further endonuclease activity was detected. Significance and Impact of the Study Restriction‐modification systems are important barriers to successful genetic modification in many bacterial species. In this study, we demonstrate an efficient and general applicable method for identifying endonuclease recognition and cleavage sites. For the study and the trails, the model organism for nitrogen fixation C lostridium pasteurianum was used. The method was proven to be reliable, and by modifying DNA at the identified sites, it is possible to prevent digestion.