REcut is an online restriction enzyme analysis tool for DNA shuffling design. It can help you choose appropriate restriction enzyme sets for optimal fragmentation of parent DNAs. It can also be used to select suitable restriction enzymes for analyzing restricted fragment length polymorphisms (RFLP) and constructing gDNA libraries. REcut is part of the online bioinformatic suite ReX. See the other tool of ReX: Xover.
Please cite: Huang W., Johnston W.A., Boden M. and Gillam E.M.J. (2016) ReX: A suite of computational tools for the design, visualization, and analysis of chimeric protein libraries. Biotechniques, 60(2):91-94. (PMID: 26842355)
Paste homologous parent DNA sequences in FASTA format.
Set the desired Minimum Fragment Length and Maximum Fragment Length. Default values are 50 bp and 400 bp, respectively.
In the first run, leave Extensive Search box unchecked. It may take several hours to finish one analysis (up to triple-enzyme combinations) . Use it as the last resort.
Alternatively, users can limit restriction enzymes to a specific set by entering up to fifty enzyme names separated by spaces. An extensive search (up to five enzymes combinations) will be performed. Users might want to save their lab RE collections in a file for quick copy & paste in future.
Submit your request and the analysis will generally be done in one minute if Extensive Search is disabled.
When analysis is done, restriction enzyme sets that can digest all parents into DNA fragments within the size limits will be displayed.
Select the Automatic Selection option and submit to see recommended restriction enzyme sets suitable for assembly by PCR. General recommended values for Min annealing length are provided in the tips section. For 80% parental sequences identity, 19 bases are good for inter-fragment self-priming.
If search doesn’t return any finding, adjust the fragment length limit settings and re-run the search. Generally, increasing upper limit would help.
Information of all restriction enzyme combinations, corresponding cleavage sites and standard deviations of fragment sizes can be downloaded and analyzed further by clicking the link provided at the bottom of result page.
Cytochrome P450 3A subfamily members 3A4, 3A5 and 3A7 are used as parents in the demonstration. After sequences submission, digestion patterns of twenty-one sets of restriction enzymes that can cleave all three parents into 50 bp to 450 bp DNA fragments are displayed like below:
To let REcut analyze what combinations of restriction enzyme sets can generate DNA fragment mix that are suitable to PCR assembly for mosaic mutants, simply use the default automatic option at the bottom of the page and submit directly. Restriction enzyme sets appropriate for PCR annealing will be displayed like below:
Two experimental setups were shown above. Each setup requires two sets of restriction enzymes, which are drawn in red and blue respectively. As shown above, there are no overlapping cleavage sites, which would be shown as red-blue crosses. You can assemble DNA fragments generated by EcoRI and SmlI mix either with fragments generated by AlWI and BtsI mix or with fragments generated by AlwI and MwoI. Both setups generate similar results and you can choose either.
If you want to select restriction enzyme sets manually and check their complementarity, you can check the boxes before enzyme names and select the manual option before submission. A digestion pattern generated by the two enzymes selected by you would be displayed as shown below:
In the example graph above, red-blue crosses appear at two positions in all parents. It means that the first set of manually selected restriction enzymes AciI and Hpy188III cleave at the same two positions as the second set AlwI and BanII do. Overlapping cleavages reduce the library diversity and can cause failure of assembly. Thus, they should be avoided. Generally, automatic selection is recommended since overlapping cleavages are checked and excluded automatically.
If your parent DNA sources are full plasmid and you only want to fragment the parent coding sequence regions and subclone chimeric open reading frames using two flanking restriction sites from the parent vector later, copy and paste the sequence from the first restriction site to the second restriction site plus flanking sequences with a length as set in the Maximum Fragment Length box at both ends. For example, if the distance between two restriction enzyme sites is 1000 bp and the upper limit of your DNA fragment is 400 bp, then you need to copy the DNA sequence from 400 bp upstream of the first restriction site to 400 bp downstream of the second restriction site. The total length of the DNA sequence to be submitted should be 1000 + 400 + 400 = 1800 bp, and that would ensure full length mosaic ORFs with two flanking restriction sites for cloning can be assembled.
If you plan to use a size-exclusion filter to separate cleavage product from unwanted oversize fragment or intact parent DNA before DNA assembly, make sure the Maximum Fragment Length is smaller than the molecular weight cut-off limit of the filter. Moreover, generally fragments too small (<30 bp) should also be avoided in case of annealing issues.
The Extensive Search will evaluate much more possible combinations of restriction enzymes, so more restriction enzyme sets applicable to all parents can be found. However, the analysis time would be much longer. At your first runs, you can try fast searches without this feature enabled. If you’re lucky, you might be able to get what you need in less than 1 minute. If no satisfying results are obtained, enabling this option will usually improve the results. It might take up to a few hours depending on the server load at that moment.
Some parents might only have very limited restriction enzyme choices. These difficult parents can be digested individually using two sets of restriction enzymes suggested in the downloadable result file. You can also temporarily exclude some parents and deal with them later using less stringent conditions.
You might want to consider increasing the Maximum Fragment Length and/or decreasing the Minimum Fragment Length slightly until applicable restriction enzyme are found. Enabling Extensive Search can also help finding more applicable enzyme sets.
Scroll up and down and observe the digestion pattern displayed. Even distribution of cleavage sites is generally preferred. When you choose the restriction enzyme sets manually, avoid choosing a set containing same restriction enzymes of the first set or having overlapped digestion sites. You can get more detailed digestion information of each restriction set by searching in the result file.
| Parental sequence identity | 50% | 60% | 70% | 80% | 90% |
| Min annealing length in bases | 30 | 25 | 21 | 19 | 17 |
You can open the result file in Excel or other spreadsheet programs. Sort the data area of each parent by the column of the standard deviation of fragment size. A smaller deviation is generally preferred for DNA shuffling. The locations of digestion sites in each parental DNA and the cleaved fragment sizes are also included for further comparison if necessary.
If you have any question or need any assistance, please don't hesitate to contact:
Your suggestions are always welcome.