Restriction Enzyme Digest Analysis
Find cut sites, fragment sizes, and gel bands for any DNA sequence with one or more restriction enzymes.
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What it does
The restriction enzyme digest analysis tool predicts where Type II restriction endonucleases cut a DNA sequence and how many fragments they produce. Paste a linear or circular sequence, pick one or more enzymes such as EcoRI, BamHI and HindIII, and the tool reports every recognition site, its position, the resulting fragment sizes, and a simulated agarose gel. It is built for molecular cloning, plasmid mapping, and verifying constructs before you order primers or run a real digest. Only standard bases A, C, G and T are read; ambiguous codes are ignored.
How it works
A restriction enzyme cuts only at its specific recognition sequence, a short palindromic site usually 4, 6 or 8 bp long. For each chosen enzyme the tool scans the DNA for every occurrence of that site and records the cut position. Fragments are then the distances between consecutive cut positions; for a circular molecule the first and last cuts wrap around, so the number of fragments equals the number of cuts, while a linear molecule produces cuts + 1 fragments when there is at least one cut. The buffer and optimal temperature for each selected enzyme are listed so you can spot incompatible conditions.
Worked example
Take the 60 bp sample plasmid and digest it with EcoRI, BamHI and HindIII. The sequence contains one EcoRI site at position 1 (GAATTC), one BamHI site at position 17 (GGATCC), and one HindIII site at position 43 (AAGCTT). On a circular plasmid these three cuts yield three fragments of roughly 16 bp, 26 bp and 18 bp, summing to the full 60 bp. On a linear molecule the same three sites produce fragments of about 16, 26 and 18 bp as well, with the ends now open. The simulated gel shows each fragment as a band positioned by size on a log scale.
When to use it
Use this tool when planning a restriction cloning step, checking whether a plasmid carries an enzyme site before inserting an insert, or confirming a PCR product will cut as expected. Pick enzymes that leave compatible sticky ends for the backbone. Watch for two pitfalls: a site may sit inside your gene of interest and destroy it, and some enzymes show star activity when glycerol or salt is off, cutting at non-specific sites. Also note that methylation in dam, dcm or CpG methylated DNA can block certain enzymes such as DpnI, so check the host strain.
Step by step
- 1
Paste your sequence
Paste or type your DNA sequence into the box; non-ACGT characters are stripped automatically.
- 2
Choose enzymes
Toggle common enzymes or search the list and select one or more restriction enzymes to digest with.
- 3
Review cuts and fragments
Read the cut table for sites and positions, then check fragment sizes and the simulated gel below.
- 4
Export or inspect
Copy fragment sizes, export a CSV, or review buffer and temperature conflicts before the real digest.
FAQ
- What does a restriction digest analysis tell me?
- It reports every recognition site for the enzymes you select, the 1-based position of each site from the 5′ end, the cuts per enzyme, and each fragment size in base pairs. For EcoRI cutting G^AATTC, a single site at position 1 on a 60 bp linear sequence yields two fragments, while the same site on a circular molecule yields one fragment spanning the origin. The simulated gel then places each fragment by size on a log scale.
- Why do circular and linear DNA produce different fragment counts?
- A cut splits a molecule into pieces at that point. A linear DNA with n cuts makes n + 1 fragments, so one cut gives two pieces. A circular plasmid has no free ends, so n cuts simply open and reseal it into n fragments; one cut yields a single linear fragment of the full length. This is why the tool asks whether your sequence is circular before computing fragment sizes.
- What is the difference between a blunt and a sticky overhang?
- An enzyme like EcoRI cuts the two strands at different offsets, leaving a single-stranded 5′ or 3′ overhang, called a sticky end, that base-pairs with a complementary overhang. A blunt cutter such as SmaI cuts both strands at the same position, leaving no overhang. Sticky ends from compatible enzymes (EcoRI and EcoRI) can be ligated, but blunt ends ligate in any orientation and need higher DNA concentration and often more ligase.
- Why would an enzyme not cut where the tool predicts?
- The tool finds sites by sequence alone, but real digests can fail for three reasons. Methylation blocks some enzymes: DpnI cuts only methylated GATC, while Dam or Dcm methylation in E. coli can protect a site. Star activity makes an enzyme cut off-site when glycerol exceeds 5% or salt is wrong. Finally, a site may hide inside secondary structure, so confirm with the conditions table first.