Restriction Mapping

Brief insight into Restriction Mapping

1. Restriction Mapping
SUNIL BHANDARI
BALKUMARI COLLEGE

2. Restriction enzymes:
•Restrictionenzymesareoneofthemostimportanttoolsin
therecombinantDNAtechnology.
•TheseareDNAcuttingenzymepresentinbacteria
(prokaryotes)thatrecognizesthespecificsitesintheDNA,
calledrestrictionsitesandmakeaDNAdoublestrandbreak
atornearrecognitionsites.
•Aftercutting,thenewlyproducedDNAendswillhaveeithera
bluntendedorsticky(staggered)end.

3. Restriction Enzyme/Endonuclease
“Also known as molecular scissor”
•Type I enzymes: They are complex multi subunit combination
restriction and modification enzymes that cut DNA at random far
from their recognition sequences
•Type II enzymes: Cut DNA at defined positions close to or within
their recognition sequences
•TypeIIIenzymes:Theycleaveoutsideoftheirrecognition
sequencesandrequiretwosuchsequencesinopposite
orientationswithinthesameDNAmoleculetoaccomplish
cleavagetheyrarelygivecompletedigests
•Type IV enzymes: Recognize modified, typically methylated DNA
and cleave at that region

4. Example of restriction enzymes and their corresponding restriction sits

5. Restriction Mapping
•RestrictionmappingisamethodusedtomapanunknownsegmentofDNA
bybreakingitintopiecesandthenidentifyingthelocationsofthe
breakpoints.Thismethodreliesupontheuseofproteinscalledrestriction
enzymes,whichcancut,ordigest,DNAmoleculesatshort,specificsequences
calledrestrictionsitesAfteraDNAsegmenthasbeendigestedusinga
restrictionenzyme,theresultingfragmentscanbeexaminedusingalaboratory
methodcalledgelelectrophoresis,whichisusedtoseparatepiecesofDNA
accordingtotheirsize.
•Onecommonmethodforconstructingarestrictionmapinvolvesdigestingthe
unknownDNAsampleinthreeways.Here,twoportionsoftheDNAsample
areindividuallydigestedwithdifferentrestrictionenzymes,andathird
portionoftheDNAsampleisdoubledigestedwithbothrestrictionenzymes
atthesametime
•Next,eachdigestionsampleisseparatedusinggelelectrophoresis,andthe
sizesoftheDNAfragmentsarerecorded.Thetotallengthofthefragmentsin
eachdigestionwillbeequalHowever,becausethelengthofeachindividual
DNAfragmentdependsuponthepositionsofitsrestrictionsites,each
restrictionsitecanbemappedaccordingtothelengthsofthefragments.

6. •Theinformationfromthedoubledigestionisparticularly
usefulforcorrectlymappingthesites.Thefinaldrawingofthe
DNAsegmentthatshowsthepositionsoftherestrictionsitesis
calledarestrictionmap
•Itispossibletodeterminethepositionofrestrictionsitesona
DNAfragmentbydigestingitwithvariousrestrictionenzymes,
singlyandincombination,andanalyzingthefragmentsizes
obtained.
•Restrictionmappingusedtobeakeyprocedureforcharacterizing
aclonedfragmentofDNA,butthisisnowmoreeasilydoneby
DNAsequencing.
•Nevertheless,analysisofrestrictionsites(orfragmentsizes)isstill
useful,forexampleincomparingthechromosomalorganizationof
differentstrains.

9. Assignment:01
Consider,a4kbDNAisdigestedwithtworestrictionenzymes(HindIIIandBamHI).The
digestedproductisrunthroughgelelectrophoresisandDNAbandsoffollowingsizeis
obtained.Drawtherestrictionmaprepresentationfollowingdigestion.
Ans:
See
last
page

10. Application of
Restriction Mapping:
1.Identification of
restriction sites
2.Insertion and deletion
study of a gene
3.Insert analysis during
cloning
4.Verification of the size
of the insert during
cloning
5.Mutation studies

11. Thank you…