Restriction-enzymes-final.ppt

Restriction enzyme

1. Restriction Enzyme

2. restriction enzymes
,Also called restriction Endonucleses
1962: “molecular scissors”discovered in in bacteria
E. coli bacteria have an enzymatic immune system that
recognizes and destroys foreign DNA
3,000 enzymes have been identified, around 200 have
unique properties, many are purified and available
commercially

3. •Molecular scissors that cut double stranded
DNA molecules at specific points.
•Found naturally in a wide variety of
prokaryotes

4. Discovery
•Arbor and Dussoix in 1962 discovered that
certain bacteria contain Endonucleases which
have the ability to cleave DNA.
•In 1970 Smith and colleagues purified and
characterized the cleavage site of a Restriction
Enzyme.
•Werner Arbor, Hamilton Smith and Daniel
Nathans shared the 1978 Nobel prize for
Medicine and Physiology for their discovery of
Restriction Enzymes

5. Restriction Enzymes
•Restrictionenzymesareendonucleases(Endo(inside),
nuclease(cutsnucleicacid),whichcatalyzethecleavage
ofthephosphodiesterbondswithinbothstrandsof
DNA.
•TheyrequireMg
+2
foractivityandgeneratea5prime
(5')phosphateanda3prime(3')hydroxylgroupatthe
pointofcleavage.

6. •Thedistinguishingfeatureofrestrictionenzymesis
thattheyonlycutatveryspecificsequencesofbases.
ThisspecificDNAsequenceiscalledrecognition
sequence.
Restrictionenzymesaretraditionallyclassifiedaccording
tothesubunitcomposition,cleavageposition,
sequence-specificityandcofactorrequirements.

7. •Arestrictionenzymerequiresaspecificdoublestranded
recognitionsequenceofnucleotidestocutDNA.
•Recognitionsitesareusually4to8basepairsinlength.
•Cleavageoccurswithinornearthesite.

8. •Restrictionenzymesarenamedaccordingtothe
organismfromwhichtheyareisolated.
•Thisisdonebyusingthefirstletterofthegenus
followedbythefirsttwolettersofthespeciesand
additionalletterornumberrepresentthestrainor
serotypes.
•Onlycertainstrainsorsub-strainsofaparticular
speciesmayproducerestrictionenzymes.
Naming of Restriction enzymes

9. Mechanism of Action
•Restriction Endonuclease scan the length of
the DNA , binds to the DNA molecule when it
recognizes a specific sequence and makes one
cut in each of the sugar phosphate backbones
of the double helix –by hydrolyzing the
phoshphodiester bond. Specifically,the bond
between the 3’ O atom and the P atom is
broken.

10. Named for bacterial genus, species, strain, and type
Example: EcoR1
Genus: Escherichia
Species: coli
Strain: R
Order discovered: 1

11. Example of RE naming:
HindII:Restriction enzyme was isolated from
Haemophilus influenzaeserotype d
•There are hundreds of different REs from different
microorganisms.
•Differentrestrictionenzymeshavedifferent
recognitionsequences.
•Thismakesitpossibletocreateawidevarietyof
differentgenefragments.

12. Examples:
Enzyme Organism from which derived
Target sequence
(cut at *)
5' -->3'
Bam HI Bacillus amyloliquefaciens G* G A T C C
Eco RI Escherichia coli RY 13 G* A A T T C
Hind III HaemophilusinflenzaeRd A* A G C T T
MboI Moraxellabovis *G A T C
PstI Providencia stuartii C T G C A * G
SmaI Serratia marcescens C CC* G GG
TaqI Thermophilus aquaticus T * C G A
XmaI Xanthamonasmalvacearum C * C CG GG

13. Restriction enzymes recognize specific 4-8 bpsequences
Some enzymes cut in a staggered fashion -“sticky ends”
EcoRI5’…GAATTC…3’
3’…CTTAAG…5’
Some enzymes cut in a direct fashion –“blunt ends”
PvuII5’…CAGCTG…3’
3’…GTCGAC…5’

17. Many recognition sequences are palindromic. For
example,
5’ GAATTC 3’
3’ CTTAAG 5’
palindromic: read the same in the opposite
direction

18. Sticky and Blunt end cutters
Not all restriction endonucleasescut symmetrically
and leave blunt ends.
Many endonucleases cleave the DNA backbones in
positions that are not directly opposite each other or
can make staggered cuts, which produce single
stranded “sticky-ends”
DNA from different sources can be spliced easily
because of these sticky-end overhangs.

19. Example of RE that produce sticky end cutters:

20. Some restriction enzymes cut DNA at opposite
base.
They leave blunt ended DNA fragments
These are called blunt end cutters
HaeIII

21. Types of Restriction Enzymes
Cleavage
site
Location of
methylase
Examples
Type I Random
Around 1000bp
away from
recognition site
Endonuclease
and methylase
located on a
single protein
molecule
EcoKI
EcoAI
CfrAI
Type II Specific
Within the
recognition site
Endonuclease
and methylase
are separate
entities
EcoRI
BamHI
Hind III
Type III Random
24-26 bp away
from recognition
site
Endonuclease
and methylase
located on a
single protein
molecule
EcoPI
HinfIII
EcoP15 I

22. Isoschizomers and
Neochischizomers
•Two Restriction enzymes that have the same recognition sequence as well
as the same cleavage site are Isoschizomers.
For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each
other
•Restriction enzymes that have the same recognition sequence but cleave
the DNA at a different site within that sequence are Neochizomers.
Eg:SmaI and XmaI
C C C G G G C C C G G G
G G G C C C G G G C C C
Xma I Sma I

23. Restriction Enzyme Uses
1.RecombinantDNAtechnology
*Discovery of enzymes that cut and paste DNA make
genetic engineering possible.
*Restriction enzyme cuts DNA and generates fragments
*Ligasejoins different DNA fragments
*DNA fragments from different species can be ligated
(joined) to create Recombinant DNA

25. 2.Cloning
Replicatesasequenceinsertedintoahost
cell
3.DNArestrictionmapping
The location of the restriction enzyme cleavage
sites on the DNA molecule.

27. •Restrictionenzymeispartofthecell’s
restriction-modificationsysteminbacteria.
•Thephenomenonofrestrictionmodificationin
bacteriaisasmallscaleimmunesystemfor
protectionfrominfectionbyforeignDNA.
•Bacteriacanprotectthemselvesonlyafterforeign
DNAhasenteredtheircytoplasm.
Biological Function

28. •Restriction Enzymes can
be used to generate a
restriction map. This
can provide useful
information in
characterizing a DNA
molecule.

29. Restriction Fragment Length
Polymorphism is a tool to study variations
among individuals & among species

30. Why don’t bacteria destroy their own DNA
with their restriction enzymes?

31. Unit Determination Assay
•Oneunitofrestrictionendonucleaseisdefined
astheamountofenzymerequiredtodigest
onemicrogramoftheappropriatesubstrate
DNAcompletelyin60minutesunderthe
conditionsspecifiedforthatenzyme.

32. Set up of a restriction enzyme reaction
•AREreactioncontainstheDNAtobe
analyzed,
•Arestrictionenzyme,
•Arestrictionenzymebuffermix.
–containsabufferingagenttomaintain
constantpH,
–andMg
++
(fromMgCl
2)asanecessary
cofactorforenzymeactivity.

33. Digestion by Restriction Enzyme
•MeasuretheDNAconcentration
–UsetheNano-dropspectrophotometertomeasure
theconcentrationofDNA,thisisusedtodetermine
theamountofHinfIrestrictionenzymetobeused.
DigestionofDNA
•Mixthefollowingcomponentsinacleanmicrotube.
•Mixgentlyandspindownforafewseconds.
•Incubateat37°Cfor16hours.