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Restriction Enzymes
Restriction Endonucleases
Cleave DNA at specific sequences
Responsible for host-controlled
restriction modification or
phenotype modification (in
bacteria)
RE PROPERTIES
Property
Type I
Type II
Type III
Restriction and
Modification
Single
multifunctional
enzyme
Separate
nuclease and
methylase
Separate
enzymes sharing
common subunit
Nuclease subunit
structure
Heterotrimer
Homodimer
Heterodimer
Cofactors
Mg2+
Mg2+ (SAM)
DNA cleavage
requirement
Two recognition
sites in any
arientation
Single recognition
site (palindrome)
Two recognition
sites in a head to
head orientation
At recognition site
At recognition site
DNA translocation
No
No
Yes
NOMENCLATURE
First letter (capital letter) first letter of the genus where
RE was isolated
Second and third letter (small letters) first two letters of
the species name (specific epithet) where RE was isolated
Fourth letter first/second letter of strain name of
organism where RE was isolated
Roman numeral number (according to order of discovery)
of RE isolated from the species.
Examples:
EcoR I Escherichia coli (RY13 strain)
Hind III Haemophilus influenzae (Rd strain)
Sma I Serratia marcescens
Taq I Thermophilus aquaticus
Kpn I Klebsiella pneumoniae
RECOGNITION SITES
Sequence specific
Variable length
Recognize mostly palindromic sequences (4-8 bp)
EcoR I - GAATTC
CTTAAG
May be interrupted by 1-9 nucleotides with no base
specificity
Sfi I - GGCCNNNNNGGCC
Some allow ambiguity in the palindrome
Acc I GTMKAC (where M = A or C and K = G or T)
Some (Type IIs) do not require palindromic sequences
Mbo II - 5.GAAGA3
RE DIGESTION ACTIVITY
ISOSCHIZOMERS
BglII
5 A-G-A-T-C-T
T-C-T-A-G-A 5
Sau3A
BamHI
5 G-A-T-C
C-T-A-G 5
5 G-G-A-T-C-C
C-C-T-A-G-G 5
STAR ACTIVITY
Altered or relaxed specificity
cleaving sequences similar (not identical) to their
defined recognition sequence
REs with known star activity:
Apo I , Ase I , BamH I, BssH II, EcoR I, EcoR V, Hind III,
Hinf I, PstI, PvuII, Sal I, Sca I, Taq I, Xmn I
Caused by:
High units to g of DNA ratio [Varies with each enzyme,
usually >100 units/g]
Low ionic strength [<25 mM]
High pH [>pH 8.0]
Presence of organic solvents [DMSO, ethanol, ethylene
glycol, dimethylacetamide, dimethylformamide,
sulphalane]
Substitution of Mg++ with other divalent cations [Mn++,
Cu++, Co++, Zn++]
RESULTS
4 bio 2
4 bio 3
4 bio 4
4 bio 5
4 bio 6
5000 bp
4969bp
3000
1000
750
500
250
Clone 2B
Clone 2A
Clone 1B
Clone 1A
5000bp
marker
Clone 2B
Clone 2A
Clone 1B
Clone 1A
5000bp
marker
5000 bp
3000
666bp
1000
750
500
250
666bp
PCR products
RE digested Plasmids using BamHI and XhoI
Verification
of fD gene insert
Plasmid pGEX-4T-1