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RESTRICTION ENZYMES

Cell and Molecular Biology Lab


Department of Biological Sciences
UST College of Science
Post Lab Discussion # 5
Restriction Enzymes
 Restriction Endonucleases
 Cleave DNA at specific
sequences
 Responsible for host-controlled
restriction modification or
phenotype modification (in
bacteria)
 growth of phages are
“restricted” to certain
strains (Luria, 1950)
 “Restriction” – bacterial
‘immunity’ against non-
self, unmethylated DNA
 Cornerstone of molecular biology
 1978 Nobel Prize in Medicine to
Werner Arber, Daniel Nathans,
Hamilton Smith
BACTERIAL RESTRICTION ENZYMES
 With two components:
 Restriction endonuclease - sequence specific nucleases
 DNA methylase – modifies DNA by methylation (prevents
cleavage of own DNA)
 > 900 known REs
 Isolated from 230 species of bacteria
RE PROPERTIES
Property Type I Type II Type III
Restriction and Single Separate Separate
Modification multifunctional nuclease and enzymes sharing
enzyme methylase common subunit
Nuclease subunit Heterotrimer Homodimer Heterodimer
structure
Cofactors ATP, Mg2+, SAM Mg2+ Mg2+ (SAM)
DNA cleavage Two recognition Single recognition Two recognition
requirement sites in any site (palindrome) sites in a head to
arientation head orientation
Site of methylation At recognition site At recognition site At recognition site

DNA translocation Yes No No


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’….GAAGA……3’
RE DIGESTION ACTIVITY
 Affedted by pH, temperature, salts and ION
concentration
 An Enzymatic Unit (u)- the amount of enzyme
required to digest 1 ug of DNA under optimal
conditions: 3-5 u/ug of genomic DNA ;1 u/ug of
plasmid DNA (Stocks typically at 10 u/ul)
ISOSCHIZOMERS

In certain cases, two


BglII 5’ A-G-A-T-C-T or more different
T-C-T-A-G-A 5’ enzymes may
recognize identical
sites

Sau3A 5’ G-A-T-C All these sticky ends


C-T-A-G 5’ are compatible

BamHI 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
 RE’s with known star activity:
 Apo I , Ase I , BamH I, BssH II, EcoR I, EcoR V, Hind III,
Hinf I, Pst I, Pvu II, Sal I, Sca I, Taq I, Xmn I
 Caused by:
 High units to µg of DNA ratio [Varies with each enzyme,
usually >100 units/µg]
 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++]
DIGESTION WITH MULTIPLE REs
 If with compatible buffers: Digest with
both enzymes in the same buffer.
 If with slightly incompatible buffers:
Cut with one enzyme, then alter the
buffer composition and cut with the
second enzyme.
 If with totally incompatible buffers:
Perform one digestion, recover the DNA
(usually by precipitation) and
resuspend in the buffer appropriate for
the second enzyme.
RESULTS
4 bio 2
4 bio 3
4 bio 4
4 bio 5
4 bio 6
Expression of Recombinant Blo t 11-fD in Escherichia
coli

Clone 1A

Clone 1B

Clone 2A

Clone 2B
Clone 1B

Clone 2B
Clone 1A

Clone 2A

5000bp
marker
5000bp
marker

5000 bp 4969bp 5000 bp

3000 3000

1000
1000
750 666bp
750 666bp 500
500 250
250

PCR products
RE digested Plasmids using BamHI and XhoI

Verification
of fD gene insert

Plasmid pGEX-4T-1