You are on page 1of 19

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 nonself, 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
Modification

Single
multifunctional
enzyme

Separate
nuclease and
methylase

Separate
enzymes sharing
common subunit

Nuclease subunit
structure

Heterotrimer

Homodimer

Heterodimer

Cofactors

ATP, Mg2+, SAM

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

Site of methylation At recognition site

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

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
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

In certain cases, two


or more different
enzymes may
recognize identical
sites
All these sticky ends
are compatible

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++]

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

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

Expression of Recombinant Blo t 11-fD in Escherichia


coli

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

You might also like