Rapid and reliable method for identification of associated endonuclease cleavage and recognition sites

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. Clostridium pasteurianum, a model organism for the stu...

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Bibliographic Details
Published in:Letters in applied microbiology 2014-06, Vol.58 (6), p.576-581
Main Authors: Jensen, T.Ø, Kvist, T, Mikkelsen, M.J, Westermann, P
Format: Article
Language:English
Subjects:
Bacteriology
Base Sequence
Binding Sites
Biological and medical sciences
Clostridium
Clostridium - genetics
Clostridium pasteurianum
Deoxyribonucleic acid
digestion
DNA
DNA Cleavage
DNA Methylation
DNA Restriction Enzymes - chemistry
DNA, Bacterial - chemistry
Fundamental and applied biological sciences. Psychology
genetic engineering
Medical research
methylation
Microbiology
nitrogen fixation
Oligonucleotides - chemistry
Plasmids
Reproducibility of Results
restriction endonucleases
Restriction Mapping
Sequence Analysis, DNA
sequencing
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Summary: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. Clostridium 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 CpaPI, which is an isoschizomer of MboI and CpaAI. Dam‐methylated DNA was used to isolate the activity of CpaAI. 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 CpaAI recognition and cleavage sites within the template sequence. By modifying DNA with both Dam and CpG methylases (M.SssI) and thereby preventing digestion by CpaPI and CpaAI, 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 Clostridium pasteurianum was used. The method was proven to be reliable, and by modifying DNA at the identified sites, it is possible to prevent digestion.
ISSN:0266-8254
1472-765X
DOI:10.1111/lam.12238