Restriction EnzymesRestriction Enzymes Aims:

Must be able to recall the basic functions of restriction enzymes.

Should be able to outline how specific restriction enzymes work.

Could be able to explain the different ends produced by restriction enzymes and their benefits.

Restriction EnzymesRestriction Enzymes RESTRICTION ENZYMES = one of the essential tools of

genetic engineering.

Purified forms of these naturally occurring bacterial enzymes are used as “molecular scalpels”.

Allow genetic engineers to cut up DNA in a controlled way.

Restriction enzymes are used to cut DNA molecules at very precise sequences of 4 to 8 base pairs called RECOGNITION SITES.

By using a ‘tool kit’ of over 400 restriction enzymes recognizing about 100 recognition sites, genetic engineers are able to isolate and sequence DNA

Restriction EnzymesRestriction Enzymes

Recognition SiteRecognition Site

GAATTC

CTTAAGDNA

CTTAAG

GAATTCcut

cut cut

restriction

enzyme

EcoRI cuts

here

Specific Recognition SequencesSpecific Recognition Sequences

Named according to the bacterial species first isolated from.

Followed by a number to distinguish different enzymes isolated from the same organism.

e.g. BamHI was isolated from the bacteria Bacillus amyloliquefaciens strain H.

A restriction enzyme cuts the double-stranded DNA molecule at its specific recognition site:

Enzyme SourceRecognition Sites

EcoRIEscherichia coli

RY13GAATTC

BamHI

Bacillus

amyloliquefaciens

H

GGATCC

HaeIIIHaemophilus

aegyptiusGGCC

HindIIIHaemophilus

influenzae RdAAGCTT

HpalHaemophilus

parainfluenzaeGTTAAC

C T T A A

A A T T C G

G

FragmentRestriction

enzyme: EcoRI

Sticky endRestriction enzyme: EcoRI

DNA from another

source

A restriction enzyme cuts the double-

stranded DNA molecule at its specific

recognition site

The two different fragments cut

by the same restriction

enzyme have identical sticky

ends and are able to join

together

The cuts produce a

DNA fragment with

two “sticky” ends

When two fragments of DNA cut by the same

restriction enzyme come together, they can join by

base-pairing

C T T A A

A A T T C

G

G A A T T C

C T T A AG

G

C T T A A

A A T T C G

G

It is possible to use restriction enzymes that cut leaving an overhang; a so-called “sticky end”.

DNA cut in such a way produces ends which may only be joined to other sticky ends with a complementary base sequence.

Sticky EndsSticky Ends

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

Blunt EndsBlunt Ends

Possible to use restriction

enzymes that cut leaving no

overhang; a so-called “blunt

end”.

DNA cut in such a way is

able to be joined to any

other blunt end fragment,

but tends to be non-specific

because there are no sticky

ends as recognition sites.

Restriction enzyme

cuts here

Recognition Site Recognition Site

DNA from another source

The cut by this type of restriction

enzyme leaves no overhang

cutcut

C C C

G G G

G G G

C C C

C C C

G G G

G G G

C C C

G G G

G G G

C C C

C C C

DNA

A special group of

enzymes can join

the pieces together

ActivityActivity

Read the information on p221 in Biozone.

Answer questions on p222 in Biozone.