Find restriction enzyme cut sites in DNA sequences using Biopython Bio.Restriction. Searches single enzymes, batches, or commercial enzyme sets and returns cut positions for linear or circular DNA. Use when locating where one or more restriction enzymes cut a sequence, screening a sequence for the presence or absence of a site, or counting how often an enzyme cuts.
Find restriction enzyme cut sites in DNA sequences using Biopython Bio.Restriction. Searches single enzymes, batches, or commercial enzyme sets and returns cut positions for linear or circular DNA. Use when locating where one or more restriction enzymes cut a sequence, screening a sequence for the presence or absence of a site, or counting how often an enzyme cuts.
tool_type
python
primary_tool
Bio.Restriction
Version Compatibility
Reference examples tested with: BioPython 1.83+ (API verified on 1.86)
Before using code patterns, verify installed versions match. If versions differ:
Python: pip show biopython then help(Bio.Restriction.Analysis) to check method names
If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying. The
Analysis "cutters" methods in particular were renamed across versions (see Common Errors).
Finding Restriction Sites
"Find where this enzyme cuts my DNA" -> Return the cut positions for one or more restriction enzymes along a linear or circular sequence.
Python: enzyme.search(seq, linear=...) for one enzyme; Bio.Restriction.Analysis(batch, seq, linear=...).full() for many.
The one fact that governs every result: search() returns a (the base immediately 3' of the cut on the top strand), the start of the recognition site. For EcoRI whose site starts at position 4, reports 5 (the base after the cut). Confusing the cut position with the recognition-site start is the single most common bug in restriction code, and it propagates silently into fragment sizes and map coordinates.
1-based position equal to the first base of the downstream fragment
not
G^AATTC
search
The Decisions That Shape A Site Search
Decision
Options
When to pick which
Enzyme scope
one enzyme / a curated RestrictionBatch / CommOnly / AllEnzymes
A named enzyme when the assay dictates it; a small batch for a cloning panel; CommOnly (623 buyable enzymes) when the answer must be an enzyme one can purchase; AllEnzymes (1088, includes non-commercial) only for exhaustive in-silico surveys
Topology
linear=True (default) / linear=False
linear=False for any plasmid, viral circle, or BAC. A circular molecule lets a site span the origin and changes fragment counts (see fragment-analysis)
Question
does it cut? / how often? / where?
search() for positions; Analysis.with_N_sites(n) for exact cut counts; bool(search()) for a yes/no screen
Use CommOnly not AllEnzymes by default: proposing an enzyme nobody sells wastes a wet-lab cycle. The README's legacy "800+" figure is stale; the installed database holds 1088 enzymes total, 623 commercially available.
Search With One Enzyme
from Bio import SeqIO
from Bio.Restriction import EcoRI
record = SeqIO.read('sequence.fasta', 'fasta')
seq = record.seq
sites = EcoRI.search(seq) # list of 1-based cut positions, e.g. [5, 14]print(f'EcoRI cuts {len(sites)} time(s) at {sites}')
ifnot sites:
print('EcoRI does not cut this sequence')
Search With A Batch Of Enzymes
Goal: Screen a sequence against several enzymes at once and keep only those that cut.
Approach: Build a RestrictionBatch, run Analysis.full() to get every enzyme's positions, then filter to cutters with with_sites().
from Bio.Restriction import RestrictionBatch, Analysis, EcoRI, BamHI, HindIII, XhoI
batch = RestrictionBatch([EcoRI, BamHI, HindIII, XhoI])
analysis = Analysis(batch, seq, linear=True)
cutters = analysis.with_sites() # {enzyme: [positions]} only enzymes that cutfor enzyme, positions in cutters.items():
print(f'{enzyme}: {positions}')
Filter By Cut Count
Goal: Separate single-cutters (linearize a plasmid), double-cutters (excise an insert), and non-cutters (safe to carry through a digest).
Approach:Analysis exposes with_N_sites(n) for an exact count and without_site() for enzymes with no site. (The older once_cutters()/twice_cutters()/only_dont_cut() names do not exist in current BioPython.)
from Bio.Restriction import Analysis, CommOnly
analysis = Analysis(CommOnly, seq, linear=False) # circular plasmid
single_cutters = analysis.with_N_sites(1) # {enzyme: [pos]} good for linearization
double_cutters = analysis.with_N_sites(2) # {enzyme: [pos, pos]} good for excision
non_cutters = analysis.without_site() # {enzyme: []} safe in a multi-step digest
all_cutters = analysis.with_sites() # any number of sitesprint(f'{len(single_cutters)} single-cutters, {len(non_cutters)} non-cutters')
# Pretty-print a chosen subset
analysis.print_as('map')
analysis.print_that(single_cutters) # formats the dict you pass it
Built-In Enzyme Collections
from Bio.Restriction import AllEnzymes, CommOnly, Analysis
print(f'{len(AllEnzymes)} known enzymes, {len(CommOnly)} commercially available')
analysis = Analysis(CommOnly, seq) # default: only buyable enzymesfor enzyme, positions in analysis.with_sites().items():
print(f'{enzyme}: {positions}')
Linear vs Circular DNA
from Bio.Restriction import EcoRI
sites_linear = EcoRI.search(seq, linear=True) # ends are free; no wrap-around
sites_circular = EcoRI.search(seq, linear=False) # a site may span the origin
A circular search can find a site that straddles position 1, which a linear search misses. Always pass linear=False for plasmids; the fragment count and map differ (see restriction-analysis/fragment-analysis).
Read An Enzyme's Cut Geometry
Goal: Know what ends an enzyme leaves before designing a ligation.
Approach:elucidate() draws the cut unambiguously; the boolean predicates and the signed ovhg summarize it. The sign convention is the trap: negative ovhg is a 5' overhang, positive is a 3' overhang, zero is blunt.
from Bio.Restriction import EcoRI, KpnI, EcoRV
for enz in (EcoRI, KpnI, EcoRV):
print(enz, enz.elucidate()) # EcoRI G^AATT_C ; KpnI G_GTAC^C ; EcoRV GAT^_ATCprint(f' site={enz.site} ovhg={enz.ovhg} ovhgseq={enz.ovhgseq!r}'f' 5prime={enz.is_5overhang()} 3prime={enz.is_3overhang()} blunt={enz.is_blunt()}')
# EcoRI.ovhg == -4 -> a 5' overhang (NOT +4). In elucidate, ^ = top-strand cut, _ = bottom-strand cut.
Not every enzyme has a fixed 6-bp palindrome. Degenerate sites use IUPAC codes (HincII GTYRAC), and interrupted palindromes carry an unspecified N spacer (BstXI CCANNNNNNTGG, DraIII CACNNNGTG). Note what is_ambiguous() actually means in BioPython: it is True when the site or cut is ambiguous -- N-spacer / interrupted sites (BstXI, DraIII) and enzymes that cut outside their site -- but it is False for a fully IUPAC-degenerate site whose cut is fixed, such as HincII GTYRAC (BioPython reports that as is_defined()). So is_ambiguous() does not detect IUPAC degeneracy; read enzyme.site for the actual letters. Either way, the expected cut frequency for a degenerate or N-containing site is not a clean 1/4^n, so do not estimate cutter rarity from site length alone for these enzymes.
from Bio.Restriction import HincII, BstXI
for enz in (HincII, BstXI):
print(enz, enz.site, 'ambiguous=', enz.is_ambiguous())
Search Many Sequences
from Bio import SeqIO
from Bio.Restriction import RestrictionBatch, Analysis, EcoRI, BamHI
batch = RestrictionBatch([EcoRI, BamHI])
for record in SeqIO.parse('sequences.fasta', 'fasta'):
cutters = Analysis(batch, record.seq).with_sites()
print(record.id, {str(e): p for e, p in cutters.items()})
Common Errors
Symptom
Cause
Fix
AttributeError: 'Analysis' object has no attribute 'once_cutters'
Method renamed across BioPython versions
Use with_N_sites(1) / with_N_sites(2); without_site() for non-cutters; with_sites() for any cutter
AttributeError: ... 'print_that_cut' / 'esite'
These names do not exist
Use print_as(...) + print_that(dct); read the cut with elucidate()
Fragment sizes or map coordinates off by a few bases
Treated search() output as the recognition-site start
The integer is the cut position = first base of the downstream fragment (1-based)
Site near the origin missed on a plasmid
Searched with linear=True
Pass linear=False for circular DNA
Reported a 5' overhang as 3' (or vice versa)
Misread the ovhg sign
Negative ovhg = 5' overhang, positive = 3' overhang, zero = blunt; confirm with elucidate()
Proposed enzyme cannot be purchased
Searched AllEnzymes
Search CommOnly when the answer must be a buyable enzyme
Related Skills
restriction-mapping - Order cut sites and draw a map with inter-site distances
enzyme-selection - Choose enzymes by cut frequency, overhang, methylation sensitivity, or compatible ends
fragment-analysis - Turn cut positions into fragment sizes and gel patterns
golden-gate-assembly - Screen a part for internal Type IIS sites before scarless assembly
sequence-io/read-sequences - Load the FASTA or GenBank sequence to search
References
Roberts RJ, Vincze T, Posfai J, Macelis D. REBASE: a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 2023;51(D1):D629-D630. doi:10.1093/nar/gkac975
Roberts RJ, Belfort M, Bestor T, et al. A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 2003;31(7):1805-1812. doi:10.1093/nar/gkg274
