BIO203 Final Term Notes

Bio203Finalterm notes · RegardsByAdmins: · VUFriends

Q1.  Asuitablesystem wasproposedbySmithandNathans(1973)

•  Thespeciesnameofhostorganismsisidentifiedbythefirstletterofgenus andfirsttwolettersofspecificepithet
•  E.coli=Eco
•  H.influenzae=Hin
•  StrainidentificationiswrittenasEcoK
•  Incase,hoststrainhasseveralrestrictionandmodificationsystems,theseare identifiedbyromannumerals,forexample,incaseofH.influenzae
•  HindI,HindII,HindIIIetc
•  AllrestrictionenzymeshavegeneralnameendonucleaseRandmodification- methylaseM followedbythesystem name,forexample,incaseofH. influenzae
•  R.HindIIIorM.HindIII

TABLE:

Enzyme Enzymesourse Recognitionsequence

SmaI Serratiamarcescens,1stenzyme CCCGGG HaeIII Haemophilusaegyptius,3rdenzyme GGCC HindIIH. influenzae,straind,2ndenzyme GTPyPuAC HindIIIH. influenzae,straind,3rdenzyme AAGCTT HamHI Bacillusamyloliquefaciens,strainH,1stenzyme GGATCCu

Targetsites:

TypeIIendonucleasesrecognizeandcleaveDNAwithinparticularsequencesof fourtoeightnucleotideswhichhaveatwofoldaxisofrotationalsymmetryi.e. referredaspalindromes 5’-GAATTC-3’ 5’-CTTAAG-3’

Q2.  Thenumberandsizeoffragmentsgeneratedbyrestrictionenzymedepend onthefrequencyofoccurrenceofthetargetsiteintheDNAtobecut

•  Fourbaserecognitionsiteoccursevery44(256)bp
•  Sixbaserecognitionsiteoccursevery46(4096)bp
•  Eightbaserecognitionsiteoccurs48(65,536)be

TABLE:

Enzyme Target Arabidopsis E.coli Human

ApaI GGGCCC 25000 15000 2000 BamHI GGATCC 6000 5000 5000 SpeI ACTAGT 8000 60000 10000etArab.

Summaryofrestrictionendonucleases:

TypeIIrestrictionenzymesareheavilyresponsibleforthecurrentexplosioninthe fieldofgenemanipulationinthattheyareessentialinformingrecombinantDNA moleculesoli

Q3. i. Nucleases

ii. DNAPolymerase iii. Reversetranscriptases iv. DNAligases (i)Nucleases:

•  NucleasesorDNasesaretheenzymesthatdegradeDNA
•  Twobroadclassesofnucleases

i).Exonucleases ii).Endonucleases

(ii)DNAPolymerase:

Anenzymethatcatalyzestemplate-dependentsynthesisofDNA

(iii)Reversetranscriptases:

AnRNA-directedDNApolymeraseinretroviruses;capableofmakingDNA complementarytoanRNA

(iv)DNAligase: Anenzymethatcreatesaphosphodiesterbondbetweenthe3’endofone DNAsegmentandthe5’of Another

Q4. MainlythreemethodsareusedforjoiningDNAinvitro (i)JoiningcovalentlyannealedcohesiveendsbyDNAligase (ii)Joiningblunt-endedfragmentsbyDNAligasefrom phageT4infectedE.coli (iiiThethirdutilizestheenzymeterminaldeoxynucleotidyltransferasetosynthesize homopolymeric3’singlestrandedtailsattheendsoffragments

Q5. E.coliandphageT4encodeenzyme,DNAligase,whichsealssinglestrandednicks betweenadjacentnucleotidesinaduplexDNAchain

•  DNAligasetocreatecovalentrecombinantDNA:

DNAligasecanrepairthenicksproducedaftertheassociationofcohesiveends ofDNAstrands.

•  AlkalinePhosphatase:
•  Anenzymeresponsibleforremovingphosphategroupsfrom manytypesof moleculesincludingDNA
•  TreatmentoflinearizedplasmidvectorDNAwithalkalinephosphataseto remove5’-terminalphosphategroupswillpreventrecircularizationand plasmiddimerformation
•  Bluntendligationvialinkermolecules:
•  JoiningDNAfragmentswithcohesiveendsbyDNAligaseisarelatively efficientprocess
•  T4DNAligasehasbeenusedtojointblunt-endedDNAmolecules
•  Adaptors:
•  Chemicallysynthesizedadaptormoleculeswhichhaveapreformedcohesive endcanbeusedtoinsertbluntendedforeignDNAintovector
•  Themaindifferencebetweenlinkerandadaptoristhatformerhavingblunt endswhilelaterhaveonecohesiveend