Bio Restriction Sites

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 1-based position equal to the first base of the downstream fragment (the base immediately 3' of the cut on the top strand), not the start of the recognition site. For EcoRI G^AATTC whose site starts at position 4, search 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.

The Decisions That Shape A Site Search

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}')
if not 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 cut
for 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 sites

print(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 enzymes
for 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^_ATC
    print(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.

Access Enzymes By Name

from Bio.Restriction import AllEnzymes

if 'EcoRI' in AllEnzymes:
    ecori = AllEnzymes.get('EcoRI')
    sites = ecori.search(seq)

Ambiguous And Interrupted Recognition Sites

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

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