Restriction Enzyme Analysis
FreeIdentify restriction enzyme cut sites and calculate fragment sizes in DNA sequences
The Restriction Enzyme Analysis tool identifies cut sites for common restriction enzymes and calculates fragment sizes in DNA sequences. This is essential for:
- **Cloning strategy design**: Planning restriction enzyme-based cloning experiments - **Plasmid mapping**: Identifying restriction sites in plasmid sequences - **Restriction mapping**: Creating restriction maps of DNA fragments - **Digestion planning**: Selecting appropriate enzymes for DNA digestion
What are restriction enzymes?** Restriction enzymes (restriction endonucleases) are proteins that cut DNA at specific recognition sequences. They are fundamental tools in molecular biology for: - Cutting DNA at specific sites - Creating compatible ends for ligation - Analyzing DNA structure and organization - Constructing recombinant DNA molecules
Enzyme types
- **Type II enzymes**: Cut at specific recognition sites (included in this tool) - **Blunt-end cutters**: Create flush ends (e.g., SmaI, EcoRV) - **Sticky-end cutters**: Create overhanging ends (e.g., EcoRI, BamHI)
Database coverage
The tool includes 20 commonly used restriction enzymes: EcoRI, BamHI, HindIII, XhoI, SalI, PstI, SmaI, KpnI, SacI, NotI, SpeI, XbaI, NcoI, BglII, ApaI, SphI, NdeI, EcoRV, HpaI, and AluI
Supported formats
- Raw DNA sequence (A, T, G, C characters) - FASTA format (with or without header) - Sequences of any length
Sequence requirements
- Must contain only valid DNA nucleotide characters: A, T, G, C - Case insensitive (both uppercase and lowercase accepted) - Ambiguity codes are accepted but may affect cut site detection
Parameters
- **Circular DNA**: Toggle for circular DNA (plasmids, vectors) - **Linear mode** (default): Treats sequence as linear DNA fragment - **Circular mode**: Treats sequence as circular DNA (plasmids), handles wraparound fragments
Enzyme selection
- By default, all 20 enzymes are analyzed - Results show cut sites for all enzymes that have recognition sites in the sequence
Example input
``` GAATTCCGGATCCATGCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGATCGAAGCTT ```
This example sequence contains: - EcoRI site (GAATTC) at the beginning - BamHI site (GGATCC) near the start - HindIII site (AAGCTT) at the end
Included Restriction Enzymes
The tool includes 20 commonly used restriction enzymes with complete information:
1. **EcoRI** - Recognition: G^AATTC, Cut type: 5' overhang (AATT) 2. **BamHI** - Recognition: G^GATCC, Cut type: 5' overhang (GATC) 3. **HindIII** - Recognition: A^AGCTT, Cut type: 5' overhang (AGCT) 4. **XhoI** - Recognition: C^TCGAG, Cut type: 5' overhang (TCGA) 5. **SalI** - Recognition: G^TCGAC, Cut type: 5' overhang (TCGA) 6. **PstI** - Recognition: CTGCA^G, Cut type: 3' overhang (TGCA) 7. **SmaI** - Recognition: CCC^GGG, Cut type: Blunt 8. **KpnI** - Recognition: GGTAC^C, Cut type: 3' overhang (GTAC) 9. **SacI** - Recognition: GAGCT^C, Cut type: 3' overhang (AGCT) 10. **NotI** - Recognition: GC^GGCCGC, Cut type: 5' overhang (GGCCGC) 11. **SpeI** - Recognition: A^CTAGT, Cut type: 5' overhang (CTAG) 12. **XbaI** - Recognition: T^CTAGA, Cut type: 5' overhang (CTAG) 13. **NcoI** - Recognition: C^CATGG, Cut type: 5' overhang (CATG) 14. **BglII** - Recognition: A^GATCT, Cut type: 5' overhang (GATC) 15. **ApaI** - Recognition: GGGCC^C, Cut type: 3' overhang (GGCC) 16. **SphI** - Recognition: GCATG^C, Cut type: 3' overhang (CATG) 17. **NdeI** - Recognition: CA^TATG, Cut type: 5' overhang (ATAT) 18. **EcoRV** - Recognition: GAT^ATC, Cut type: Blunt 19. **HpaI** - Recognition: GTT^AAC, Cut type: Blunt 20. **AluI** - Recognition: AG^CT, Cut type: Blunt
Enzyme information includes
- Recognition sequence (the specific DNA sequence recognized) - Cut position (where the enzyme cuts within the recognition site) - Cut type (blunt or sticky end, overhang direction) - Overhang sequence (for sticky-end enzymes)
Cut Site Results
For each enzyme that has recognition sites in your sequence, the tool provides:
- **Enzyme name**: Name of the restriction enzyme - **Recognition site**: The specific DNA sequence recognized by the enzyme - **Cut position**: Position where the enzyme cuts (1-based coordinates) - **Cut type**: Blunt or sticky end (with overhang direction if applicable) - **Overhang**: The overhang sequence for sticky-end enzymes (e.g., "AATT" for EcoRI)
Fragment Analysis
After identifying all cut sites, the tool calculates fragments:
- **Fragment number**: Sequential numbering of fragments - **Start position**: Starting position of the fragment (1-based) - **End position**: Ending position of the fragment (1-based) - **Length**: Fragment size in base pairs - **Sequence**: Complete nucleotide sequence of the fragment
Summary Statistics
- **Total cut sites**: Total number of recognition sites found across all enzymes - **Total fragments**: Number of fragments created by all cuts - **Sequence length**: Total length of input sequence
Circular DNA handling
When circular DNA mode is enabled: - Wraparound fragments are correctly identified (fragments spanning the origin) - Fragment positions account for circular topology - Fragment sizes are calculated correctly for circular molecules
**1. Cloning Strategy Design** - Select appropriate restriction enzymes for cloning - Design compatible ends for ligation - Plan multi-step cloning strategies - Verify restriction sites in cloning vectors
**2. Plasmid Analysis and Mapping** - Create restriction maps of plasmids - Verify plasmid identity and structure - Identify suitable cloning sites - Plan plasmid modifications and insertions
**3. DNA Fragment Analysis** - Analyze restriction patterns of DNA fragments - Verify fragment sizes after digestion - Plan gel electrophoresis experiments - Design restriction-based genotyping assays
**4. Recombinant DNA Construction** - Design restriction-based cloning strategies - Select enzymes for directional cloning - Plan insert preparation and vector linearization - Verify compatibility of restriction sites
**5. Molecular Biology Experiments** - Plan restriction digestions - Select enzymes for DNA analysis - Design restriction-based assays - Verify experimental results
1. **Choose appropriate enzymes**: - Select enzymes with compatible overhangs for ligation - Avoid enzymes that cut within your insert sequence - Consider enzyme availability and cost
2. **Understand sticky vs. blunt ends**: - Sticky ends (overhangs) allow directional cloning and more efficient ligation - Blunt ends are less specific but work with any compatible blunt end - Compatible sticky ends (same overhang sequence) can be ligated together
3. **Circular DNA considerations**: - Always use circular mode for plasmids and vectors - Circular mode correctly handles cuts that span the origin - Wraparound fragments are properly calculated in circular mode
4. **Fragment size planning**: - Consider gel resolution when planning digestions - Smaller fragments (< 100 bp) may be difficult to visualize - Larger fragments (> 10 kb) may require special gel conditions
5. **Multi-enzyme digests**: - Check compatibility of enzyme buffers - Some enzymes require specific buffer conditions - Consider sequential digestions if enzymes are incompatible
6. **Experimental validation**: - Always verify predicted cut sites experimentally - Check fragment sizes on agarose gels - Confirm enzyme activity and specificity
7. **Cloning considerations**: - Ensure insert and vector have compatible ends - Avoid internal restriction sites in inserts - Consider using multiple enzymes for directional cloning