The program accepts an input sequence, which can be pasted in, picked up from a local file or retrieved from NCBI as a Genbank file via its accession number. It calculates all long open reading frames and then displays the sequence, the long ORFs and all NEB enzymes that cut it just once. It also shows the enzymes which could be used in a complete digest to excise each open reading frame that it finds. From the initial display there are options to go further, including custom digestion with enzymes of your choice and various displays of the digest. One feature that is implemented on some pages is that moving the mouse over an enzyme name will produce a box with the recognition sequence and will also underline the bases of that recognition within the display. On the digest, moving the mouse on to the bands will display the fragment length and the enzyme that produced each end of the fragment.
Ambiguous nucleotides are allowed in the input sequence, up to 5 in any 20 nt window. If there are more then a warning message is displayed and all degenerate bases are ignored in the sequence. Recognition sites which depend on the precise sequence but overlap a degenerate base are marked by putting the enzyme name in parentheses.
When the program searches for ORFs, by default it uses the bacterial genetic code (which can be changed on the opening page) to identify start and stop codons. If two ORFs significantly overlap then the smaller one is dropped, keeping only the longest, non-overlapping ones. Since not only ATG is treated as an initiation codon, some ORFs may be predicted to be longer than they actually are. In this case you can either choose the "Standard" genetic code, which has less alternative start codons, or use the "edit ORF" feature to specify the correct coordinates.
NEBcutter incorporates everything that is known about the methylation sensitivity of any of the enzymes displayed when they overlap say a Dam site or a Dcm site as well as CpG, EcoK and EcoB sites. When theoretical digests are performed the results include a comparison of the effects of methylation, highlighting the bands that shift.
We have tried to make most of the options intuitive with buttons to provide obvious analyses. Please feel free to give us feedback by using the "comment" feature in the top right hand corner of each page.
Citing NEBcutter
Tamas Vincze and Richard J. Roberts