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Restriction Enzymes and Their Applications

Here are some guidelines for selecting restriction enzymes: - Choose enzymes with 4-6 bp recognition sites for frequent cutting of genomic DNA. Longer sites cut less frequently. - Consider enzymes that leave compatible cohesive ends for subsequent cloning. Blunt ends can be used but are less efficient. - Select enzymes that don't cut within the region of interest to avoid undesirable fragmentation. - Consult catalogs for enzyme information like recognition sites, suppliers, prices to find ones suitable for your application and budget. - Common workhorse enzymes like EcoRI, HindIII, BamHI are good starters due to widespread availability and experience using them.

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Faisal Akbar
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37 views27 pages

Restriction Enzymes and Their Applications

Here are some guidelines for selecting restriction enzymes: - Choose enzymes with 4-6 bp recognition sites for frequent cutting of genomic DNA. Longer sites cut less frequently. - Consider enzymes that leave compatible cohesive ends for subsequent cloning. Blunt ends can be used but are less efficient. - Select enzymes that don't cut within the region of interest to avoid undesirable fragmentation. - Consult catalogs for enzyme information like recognition sites, suppliers, prices to find ones suitable for your application and budget. - Common workhorse enzymes like EcoRI, HindIII, BamHI are good starters due to widespread availability and experience using them.

Uploaded by

Faisal Akbar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Restriction

est ct o e endonucleases
do uc eases
and their applications
History
Hi t off restriction
t i ti
endonucleases
e do uc eases a andd its
ts role
oe
in establishing molecular
bi l
biology
Restriction enzymes
• Over 10,000 bacteria species have been
screened for restriction enzymes
• Over
O 2,500
2 500 restriction
t i ti enzymes have
h been
b found
f d
• Over 250 distinct specificities
• Occasionally enzymes with novel DNA sequence
specificities are still found while most now prove
to be duplicates (isoschizomers) of already
di
discovered d specificities.
ifi iti
Restriction Enzyme Function
• It is generally believed that the biological
function of restriction enzymes is to
protect cells from foreign DNA.

• Infecting DNA is cleaved (restricted) by


the restriction enzyme(s) preventing it
f
from successfully
f ll replicating
li ti and d
parasitizing the cell.
Why the bacteria does not kill itself?
The Restriction Enzyme Modification Systems

if everything gets cleaved, how come the bacteria


does not kill itself?

• Usually, organisms that make restriction enzymes


also make a companion modification enzyme
((DNA methyltransferase)
y ) that p
protects their own
DNA from cleavage.

• These enzymes
y recognize
g the same DNA
sequence as the restriction enzyme they
accompany, but instead of cleaving the sequence,
they disguise it by methylating one of the bases in
each
h DNA strand.
t d
Classification of Restriction
enzymes
Class I Class II (93%) Class III

Restriction-methylase Homo-dimers, Restriction-methylase


on the same subunit methylase on a on the same subunit
separate
p subunit

ATP-dependent Mg++ dependent ATP-dependent

Binds to DNA recognize symmetric Cut the DNA at the


recognition site and DNA sequences and recognition site and
cuts
t DNA randomly
d l - cleave
l within
ithi the
th th di
then dissociate
i t from
f
any DNA as long as it sequences the DNA
comes in contact
Type II Restriction enzymes
are endonucleases
that cut DNA at specific sites, and are most
useful for molecular biology research
Type II Restriction enzymes
Recognition sites
are
P li d i
Palindroimes:
121
IFFI ABA
IFFI,
AAGCTT
TTCGAA
How do I know what sequence
each enzyme cut?

• Test by
y cutting
g DNA of known sequence
q

• Commercial sources are tested already,


y,
and you find a catalog
Some popular Biotechnology Companies

• Life Technologies (BRL/GIBCO)


• New England Biolabs
• Amersham Pharmacia Biotech
• Qiagen
• P
Promega
• Clonetech
• Invitrogen
• Stratagene
• ...
Nomenclature of restriction enzyme

• Eco R1: E coli


• Pst I: Providencia stuartii
• Hind III: Haemophilus influenza
• Not I: Norcardia otitidis-caviarum

• What do you name a restriction enzyme


isolated from Xanthomonas graminis?
How long is the recognition sequence

• 4 bp: e.g., Taq 1, HpaII, MspI

• 6 bp: e.g., EcoR1, HindIII, BamH1, PstI, salI

• 8 bp: Not I, Sfi I


Recognition sequence may be
interrupted or ambiguous

Acc I: GT(at/gc)AC
( g )

Bgl
g I: GCCNNNNNGGC

Afl III: ACPuPyGT


Three types of ends produced
by type II restriction enzymes

• 3’-overhang (protruding)
• 5’-overhang
5 overhang
• Blunt end
5’-overhang
EcoR I
5’-----------------------gaattc---------------------------3’
5 -----------------------gaattc---------------------------3
3’-----------------------cttaag--------------------------5’

X EcoR1

5’-----------------------g
g aattc
aattc---------------------------3’
3
3’-----------------------cttaa +
g---------------------------5’
3’-overhang
Pst I:
5’-----------------------ctgcag---------------------------3’
5 -----------------------ctgcag---------------------------3
3’-----------------------gacgtc--------------------------5’

X PstI

5’-----------------------ctgca-3’
g 5’-g---------------------------3’
5 g 3
3’-----------------------g-5’ +
3’- actgc---------------------------5’
Blunt end
EcoR V
5’-----------------------gatatc---------------------------3’
5 -----------------------gatatc---------------------------3
3’-----------------------ctatag--------------------------5’

X EcoR V

5’-----------------------gat
g
+ atc---------------------------3’
atc 3
3’-----------------------cta tag---------------------------5’
Only Compatible,
base-pairable ends
can ligate
li t
Odds of cutting at a segment of DNA

• 4 bp cutter: 44 = 256 bp
p cutter: 46 = 4 kb
• 6 bp
• 8 bp cutter: 48 = 64 kb

• ??? How many do you predict Eco R1 to


genome of 8 x 109 bp
cut catfish g p
What do you expect if
you digest with your
plasmid DNA with 4-
p
bp cutters
What do you expect if
you digest with your
plasmid DNA with 6-
p
bp cutters
What do you expect if
you digest with your
plasmid DNA with 8-
p
bp cutters
Restriction mapping
EcoR1

EcoR1

EcoR1
Pst I

Pst I
E

E
P

P
0.6 kb 2.4 kb 6.0 kb 1.0 kb
7.7 kb
1.5 kb 0.8 kb

10 kb

What do you expect to see with double digest,


if reaction is complete?
0.6 kb, 0.9 kb*, 1.5 kb, 6.0 kb, 0.2 kb, 0.8 kb.
What do you expect if
you digest with your
genomic DNA with 4-
g
bp cutters
What do you expect if
you digest with your
genomic DNA with 6-
g
bp cutters
What do you expect if
you digest with your
genomic DNA with 8-
g
bp cutters
Selection of restriction
enzymes
Of Over 250 commercially available and
over 2,000 total, how do I know what to
use?
• Cutting frequency
• Easy to work with
• Economical

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