Restriction Digestion

This presentation contains information about restriction enzymes, its nomenclature, restriction digestion, and its application. This also contains information about the chemicals used in restriction and also explains the general procedure of restriction digestion of DNA

1. Restriction Digestion
By-
Meenakshi Das

2. INTRODUCTION
❖Genemanipulationrequires
enzymestocutDNAmolecules.
❖Therestrictionendonucleasescutat
theinteriorpartofDNA.
❖Theseenzymesarefoundinbacteria
andinvivoareinvolvedin
recognitionanddestructionofforeign
DNA.
❖InvadingphageDNAforinstance
willberestrictedbysuchenzymes.
❖Thebacteriaprotecttheirown
DNAbymodificationprocess.
Bacterial
Cell
Bacterial DNA
Bacteriophage
Phage DNA
Restriction Enzyme
Digested
DNA
Restriction Digestion:
Bacterial innate Immune System
http://www.aun.edu.eg/molecular_biology/gene&protein/2nd%20day/5%206%20restriction%20enzymes%20and%20ligases%20[Compatibility%20M
ode].pdf

3. ❖The essential feature of restriction endonucleases is that
these enzymes recognize a particular sequence ofbases.
❖Type II restriction enzymes cut the DNA within the
recognizedsequences.
❖Each enzyme has its own characteristic recognition
sequence and it may be 4 to 7bases long with dyad
symmetry.
❖With the availability of nearly 50 restriction enzymes
commercially, it is now possible to construct the physical map
of genes after digestion of the DNA with different restriction
enzymes and subsequent of DNA fragments on agarosegel.

4. PRINCIPLE
❖RestrictionenzymeEcoRIis
isolatedfromE.coli
❖EcoRIrecognizesandcleavesthe
sequence5”-GAATTC-3”
❖EcoRIgeneratescohesiveor
stickyends
❖HindIII isolatedfrom
Haemophilusinfluenza
❖Itcleavesthesequence5”-
AAGCTT-3”
❖HindIIIgeneratescohesiveor
stickyends.
EcoRI
HindIII
G|AATTC
CTTAA|G
A|AGCTT
TTCGA|A
Recognition sequence and cutting
sites are shown by“|”and

5. NOMENCLATURE OF RE
EcoRI
E =Escherichia
co = coli
R = strainRY12
I= Roman numeral one
genusname
speciesname
strain
FirstEnzymeisolated
fromthisspecies
HindIII
H= Haemophilus
in =influenzae
d =serotyped
III = Roman numeral Three
genusname
speciesname
serotype
ThirdEnzymeisolated
fromthisspecies

6. TYPES OF RESTRICTIONENZYME
Three types of
restriction enzyme
❖TYPEI
❖TYPEII
❖TYPEIII
Restriction
Enzymes
Cleavage sites
Location of
Methylase
Common
Examples
TYPEI
Random
Away from
recognition site
Present with
Endonuclease
on a single
protein
EcoKI
EcoAI
CfrAI
TYPEII
Very specific
Within the
recognition site
Separate
proteins
EcoRI
BamHI
HindIII
TYPEIII
Random
Away from
recognition site
Present with
Endonuclease
on a single
protein
EcoPI
HinfIII
EcoP15I

7. TYPE IRE
❖They are complex enzymes which act as endonuclease and methylase
function
❖They requireATP,Mg+2
❖They are single, multi-functional enzyme which recognizes 15bp in
length and cleavage site is1000bp
❖They show specificity for recognition but not for cleaveage
❖They produce heterogeneousfragments
❖Not used in gene cloningtechniques
❖Examples:EcoK,EcoB

8. TYPE IIRE
❖Most common RE used in genecloning
❖They are simple enzyme having singlepolypeptide
❖They have separate methylase and endonuclease activity
❖The recognition and cutting site is the sameone
❖They generally recognize six nucleotide
❖Someenzymesalso recognize 4,5,or 8bp
❖Examples: PvuI,PvuII,EcoRI

9. Cutting site of RE TypeII
The recognition sequences for Type II RE form
palindromes with rotational symmetry .
GAA TTC
CTT AAG
EcoRIrecognition site

10. Cutting site of RE TypeII
Cleave DNA to generate different“ends”
❖Staggeredcut
❖Bluntend

11. TYPEIIIRE
❖There are twosubunits
❖One is for recognition and modification anotherfor nuclease activity.
❖They requireATPand Mg
2+
❖The problem is that the recognition sitesare assymetricnon-palindromic
❖The single strand ends produce by TYPEIII differ from each other and cannot recombine at
random
❖They lack ATPase activity
❖Example: HgaI,MboII

12. How to do Restriction Digestion??
Requirements for Restriction Digestion
Need for Restriction Digestion

13. MATERIALSREQUIRED
❖Isolated DNA
❖Restriction Buffer (10x) Sodiumacetate
❖Ethanol
❖Sterile double distilledwater
❖Restrictionenzymes
❖Agarose
❖Gel running buffer
❖Ethidium Bromide
❖Agarose gel electrophoresis unit
❖Micropipette
❖Microcentrifugetubes
❖DNAmarker
❖bucket with crushedice,
❖pipettetips
Other requirements:

14. Preparation for Restriction Digestion
Recipes(μl) 1 2 3 5
DistilledWater 16 16 14 14
10xbuffer 2 2 2 2
MarkerDNA 2 - - -
SampleDNA - 2 2 2
HindIII - - 2 -
BamHI - - - 2
Totalvolume
(μl)
20 20 20 20

15. Procedure
1.Label microfuge tubes 1 to 4and arrangethem open in arack.
2.Bring all reactants on ice.
3.DNA samples stored frozen are thawed quickly and brought onice.
4.Prepare the following reaction mixes by carefully pipetting into the bottom of
microfugetubes.
5.Fasten the cap on each tube.
6.Mix contents of tubes carefully.
7.Centrifuge all the microfuge tubes for 2 second to settle the contents at the bottom
oftubes.
8.Incubate 30
OC in water bath for 60 minutesor longer ifnecessary.

16. 9.To the digested reaction mixture, add 2 μl of 3 M sodium
acetate and 50 μl absolute ethanol for precipitation. Keep it
at -20
O
C for 20minutes.
10.Recover the DNA by centrifugation for 15 minutes.
11.Wash the pellets twice with 70 % ethanol.
Remove supernatant.
12.Air dry thepellets.Dissolve the pellets in 20 μl
TE buffer and store at-40
O
C
13.The digested DNA fragments can be separated
throughelectrophoresis.

18. ❖Restrictionenzymedigestionisacommonlyusedtechniquefor
molecularcloning
❖Itisalsousedtoquicklychecktheidentityofaplasmidbydiagnostic
digest
❖Studyofmutationandpopulation-widepolymorphism
❖UsedinLinkageMapping
❖UsedinDNAprobepreparation
❖DNAfingerprintingandPaternitytest(RFLPMethod).RFLPstandsfor
RestrictionFragmentLengthPolymorphism
❖PreparationofExpressionVector
Application

19. PRECAUTION
Makesurethattherestrictionenzymedoesnotexceedmore
than10%ofthetotalreactionvolume,otherwisethe
glycerolandtheEDTAintheenzymestoragebuffermay
inhibitdigestionprocess.