STRUCTURE:
10001000000bases/gene,
humangenome3billionbases,20,000genes,23pairsofchromosomesonchromo,p=small,q=big
REPLICATION
exonuclease=repairenzyme
discontinuousleadingvs.lagging(Okazaki)
1953Watson,Crick,Wilkins,Franklin=structure
GENETICS
meiosis2,4haploids,indepassort
Xlinked(sex)INMALES:traitscolorblind,hemophilia,ALD,MD,malepatternbaldness
Karyotypenondisjunctiondisorders:Klinefelters(XXY)Turner(Xo)Down(tri21)
diploid,monosomy,trisomy
GENEEXPRESSION
operonslac/trpJacobandMonod
1.regulatorgeneproducingtherepressormolecule
2.operatorgenerepressorbindshere.Repressibleitbinds,Inducibleitreleases.RNApolymerasebindshere
3.structuralgeneisresponsiblefortheproductionofenzymesusedinbiochemicalpathways
4.repressormoleculeisresponsibleforcombiningtoeitherlactoseortheoperatorgene.
DETECTIONofgeneexpressionBLOTTINGSNoWDRoP
MUTATIONSchromo#nondisjunction,chromossizeadd/sub/invert.genesframeshift=add/sub,point=
substitution,
SEQUENCINGSangerddNTPareNOTelongating.Endgrowth,runongel
RFLPRestrictionfragmentlengthpolymorphismexploitsvariationsinhomologousDNAsequencesDNAbroken
intopiecesbyrestrictionenzymes>restrictionfragmentsareseparatedbylengthbygelelectrophoresis
Plasmidselectionandisolationmini,midi,maxiprep(culturevolume>plasmidyeild)anextractionwBasic
buffer
selectionwdrug(amp^r)
PCRamplifyDNAcopiesexponentially,regionbetweenprimers,
forIDordetectionofdisease,TaqDNApolymerase,Thermocycler(denature,anneal,extendbytemp95,50,72o)
KaryMullis1983Nobel
Polymerasechainreaction(PCR)
oCopyagenemanytimestogeneratesufficientamountofmaterialforexperiments
oDescribePCRcycle:denaturation,annealingofprimers,primerextension
oReagents:DNApolymerase,freenucleotides
oNumberofDNAcopiesdoublesineachcycle(chainreaction)
ELISAEnzymeLinkedImmunoSorbentAssayidentifyantibody/antigenwiththeother,colorchangingenzyme
linkedto2ndaryantibody
GENETHERAPYsupplementoraltergenestotreatdisease.Replaceamutatedgene,viralvectors,Cystic
FibrosisplaceCFTRgenewhichregulatessaltsintotheColdvirusorliposome.
Ingenetics,andoperonisafunctioningunitofgenomicDNAcontainingaclusterofgenesunderthecontrolofa
singleregulatorysignalorpromoter.
lacmetabolismoflactoseinbacteria,esp.E.colitrpcodesfortryptophanproduction
Thelacoperonconsistsofthreestructuralgenes,andapromoter,aterminator,regulator,andanoperator.The
threestructuralgenesare:lacZ,lacY,andlacA.
lacZencodesgalactosidase(LacZ),anintracellularenzymethatcleavesthedisaccharidelactoseintoglucose
andgalactose.
lacYencodesgalactosidepermease(LacY),amembraneboundtransportproteinthatpumpslactoseintothe
cell.
lacAencodesgalactosidetransacetylase(LacA),anenzymethattransfersanacetylgroupfromacetylCoAto
galactosides.
OnlylacZandlacYappeartobenecessaryforlactosecatabolism.
TRP:negativerepressivefeedbackmechanism.TherepressorforthetrpoperonproducedupstreambytrpR
gene,whichiscontinuallyexpressedatalowlevel.Createsmonomers,whichassociateintotetramers.These
tetramersareinactive,aredissolvedinnucleoplasm.Whentryptophanispresent,thesetryptophanrepressor
tetramersbindtotryptophan,causechangeinconformation(inrepressor),allowsrepressortotheoperator.This
preventsRNApolymerasefrombindingtoandtranscribingtheoperon,sotryptophanisnotproducedfromits
precursor.Whentryptophanisnotpresent,therepressorisinitsinactiveconformationandcannotbindthe
operatorregion,sotranscriptionisnotinhibitedbytherepressor.
Autoradiography:TheprocessofdetectionofradioactivelylabeledmoleculesbyexposureofanXraysensitivefilm.
Hybridization:annealingofasinglestrandedDNAtoitscomplimentaryregiononanothersinglestrandedDNA
ShorttandemrepeatsorSTRsarenoncodingDNAthatconsistof25nucleotidesequencesrepeatedmanytimes(intandem).
Theirnumberandarrangementinhumanchromosomesisuniquetoeachindividual.
DNASequencing
oDideoxyDNAsequencing(FrederickSanger):Determinenucleotidesequencefromsequencinggel
Standarddeoxynucleotides(dATP,dGTP,dCTPanddTTP)
Chainterminatingdideoxynucleotides(ddATP,ddGTP,ddCTP,orddTTP)lackthe3OHgroup
TheDNAiintofourseparatesequencingreactions,containingallfourofthestandarddeoxynucleotidesandDNApoly.Toeach
reactionisaddedonlyoneofthefourdideoxynucleotides..FollowingroundsoftemplateDNAextensionfromtheboundprimer,
theresultingDNAfragmentsareheatdenaturedandseparatedbysizeusinggelelectrophoresis.Thisisfrequentlyperformed
usingadenaturingpolyacrylamideureagelwitheachofthefourreactionsruninoneoffourindividuallanes(lanesA,T,G,C).
TheDNAbandsmaythenbevisualizedbyautoradiographyorUVlightandtheDNAsequencecanbedirectlyreadofftheXray
filmorgelimage.
