Topic 3 Biochemical Analytical Technique Part-2 Flashcards
what is dna?
Deoxyribonucleicacidcontain genetic material in its nucleus and contains 23 pairs of chr
what are the monomeric units of DNA and RNA respectively?
deoxyribonucleotidesand
ribonucleotides.
what are the 3 components of DNA?
a)Nitrogenousheterocyclicbase
(purineorpyrimidine)
b) Pentosesugar
c) Phosphategroup
what are the 4 types of bases in DNA?
Adenine,Thymine,
Cytosine, Guanine
AdeninelinkswithThymine CytosinelinkswithGuanine
what are the 2 diff between dna and rna
DNA building block is deoxyribonucleotides while RNA is ribonucleotides
DNA function is for storing and passing down genetic information while RNA converts codes into proteins to carry out cellular functions
Sugar in DNA is deoxyribose sugar while RNA is ribose sugar
bases in DNA Is adenine, thymine, guanine and cytosine.
bases in RNA is adenine, uracil, guanine and cytosine
what is a protein?
Madeofaminoacids Containbothacidiccarboxyl(‐COOH)and basicamino(‐NH2) groupattachedtoαcarbonatom. DifferwithrespecttoRgroup.
how is a protein formed?
Formedbycondensationofaminoacids which form a chain aka polypeptide. 2 or more polypeptides together may acquire a functional form and a 3D structure to become a protein. Proteins are long chains of Aas held together by peptide bonds
**Polypeptideshavemolecularweightof1500orless.Proteinshavemolecularweightof5000ormore.
what is a Zwitterionic aminoacids?
Whendissolveinwater
theycontainanaminegroup(basic)andacarboxylicgroup(acidic)
Thenetchargeoftheentiremoleculeiszero.
how to calculate isoelectric pH?
IsoelectricpH=(pK1 +pK2 )/2
what is the isoelectronic pH?
MoleculehasnonetelectricchargeatthispHandwillnotmoveinanelectricfield
aminoacidhastheleastbufferingeffect (no ability to change the pH of a system)
what are some examples where DNA can be collected?
Couldbealickedenvelope,dirtylaundry,acigarettebuttorsalivaetc.
what are some Specialprecautionswe should take whenhandlingspecimens:
gloves,disposableinstruments,avoidtalkingandsneezing,avoidtouchingsamplewithyourskin,air‐drytheevidencebeforepackagingsomolddoesnotgrow
what degrades evidence?
sunlight,hightemperatures,bacteria,
moisture
what is the ideal sample?
e:1mLoffresh,wholeblood(whitebloodcells)treatedwithEDTA
**Ethylenediaminetetraaceticacid(EDTA)stronglyandirreversiblychelates(binds)calciumions,preventingbloodfromclotting
what are the 3 bioanalytical techniques for DNA typing?
PolymeraseChainReaction(PCR)
RestrictionFragmentLengthPolymorphisms(RFLP)
electrophoresis
what is the function for PCR?
ToamplifyminutequantitiesofDNAespeciallywhenmoreDNAtemplateisrequiredforfurtherreactions/analysis
what is the function for RFLP?
TofragmentDNAsamplesintopieces,utilizeshomologousDNAdifferenceswithinrestrictionenzymebindingsites.
what is the function for electrophoresis?
Toseparatethefragmentsbasedonchargedandlength/size.
what is the PCR test mainly for? what is its main advantage?
‐Techniqueformakingcopiesoramplifyinga
specificsequenceofDNAinashortperiodoftime.
‐CellfreeDNAamplification
ConvenientmethodforobtaininglargenumberofDNAcopies.Canbethoughtofasamolecularphotocopier.
advantage:
RobustandworksforsmallamountsofDNAandworkseveniftheDNAisbadlydegraded.
what are the basic steps of PCR? (DAE)
- •Denaturation:
–DNAfragmentsareheatedathightemperatures(94oC)–DNAdoublehelixreducedtosinglestrands. - •Annealing:
–Thereactionmixtureiscooleddown.
–PrimersannealtothecomplementaryregionsintheDNAtemplatestrands
–Doublestrandsareformedagain. - Extension: TheDNApolymerasesynthesizesthecompletecomplementarystrand.
how do we calculate the number of DNA produced in a PCR cycle?
No of DNA produced = 2^n
n = no of cycles
what is electrophoresis?
separation method based on the differential rate of migration of charged species (DNA fragments and other macromolecules) in a buffer solution across which has been applied a dc electric field.
what is the rate of migration of a given species dependent on?
dependsuponcharge and sizeofions.
what is the unique ability of electrophoresis? what is it commonly used for?
•Uniqueabilitytoseparatechargedmacromolecules ofinterest. •PopularforanalysisofDNA,RNAandprotein
what are the 3 principles of separation of DNA fragments in electrophoresis?
Formofseparationusedtoidentifymacromoleculesofbiologicalcompoundse.g. DNAandproteins
Movementofchargedparticlesinanelectricfield.Anegativelychargedparticlewillmovetowardthepositiveendandviceversa.ProteinsarechargedatallpHexceptattheirisoelectricpoint(pI).
Differencesinsizeandchargebetweenproteinsresultsindifferentmigrationrateswhentheyareinanelectricfield.
what are the 2 apparatus used in electrophoresis?
(a) D.C.powersupplywithcontrolforbothvoltageandcurrentoutput.
(b) Electrophoresisunitcontainingelectrodes,buffersreservoirandasolidsupportforelectrophoresismedium.
explain the general principles of electrophoresis technique in terms of zwitterions, cations and anions
Zwitterion–remains stagnant, a molecule that contains an equal number of positively-and negatively-charged functional groups. Thus overall is neutral.
Cation-a positively charged ion, i.e.one that would be attracted to the cathode
Anion–a negatively charged ion, i.e.one that would be attracted to the anode
Smaller molecules travel longer distances while heavier molecules travel shorter distances
what are the 2 types of electrophoresis?
a) Gelelectrophoresis(Supportingmedium:Gel):
(i) Starch
(ii) Agarose(Agarosegelelectrophoresis)
(iii) Polyacrylamide(SodiumDodecylSulphate–PolyAcrylamide Gel Electrophoresis)
b)Capillaryelectrophoresis(Supportingmedium:Capillary)
briefly describe starch as a supporting medium for electrophoresis. what is its application, advantage and disadvantage?
Starchassupportingmedium: Preparebyheatingandcoolingmixtureofpartiallyhydrolyzed starchinanappropriatebuffer.
Disadvantage:
(1) Nowaytodetermineexactporesizeofstarchgels.
(2) Differentbatchesareinconsistentwithrespecttoporesizeforsamepercentageofstarch.
Advantage: Easeofapplyinghistochemicaltests.
Application: Analysisofisoenzyme patterns.
briefly describe agarose gel as a supporting medium for electrophoresis.
SeparationofDNAfragments>1000basepairs.
AllDNAisofuniformshapeandcharge‐massratio.
Separatedbymolecularweight/sizeandmeasuredasbasepairs.
what are the steps involved in agarose gel electrophoresis?
•Sampleisappliedasaspotonawellinsupportingmediumofelectrophoresiskit.
•ApplyD.C.andtheionsofthesamplemigratetowardstheelectrodesofoppositepolarityatcharacteristicratestoformbands
•Migrationrateisdependentonchargeandoncompletionofelectrophoretogram,thesupportingmediumisdriedandsamplecomponentsaredetected
•Proteinmoleculescanbeseparatedfromeachotherbytakingadvantageoftheiroverallcharged(positiveornegative).
•Inelectricfieldbetweentwoelectrodes,apositivelychargedparticlewillmovetowardnegativeelectrodeandanegativelychargedparticlewillmovetowardpositiveelectrode.
briefly describe polyacrylamide gel as a supporting medium for electrophoresis.
Cross‐linkedpolymer:
suppressconvectivecurrentsproducedbysmalltemperature
gradients,arequirementforeffectiveseparation.
•serveasmolecularsievesthatenhanceseparation.
why is polyacrylamide a commonly used gel medium?
(a) chemicallyinert
(b) readilyformedbypolymerisationofacrylamide.
(c) Poresizecontrolledbychoosingvariousconcentrationsofacrylamide(0.7–2%).
(d) highdegreeofreproducibility&resolution
describe how the qualitative results of gel electrophoresis comes about
Mobilityofproteinsislinearlyproportionaltothelogarithmoftheirmass.
what is the main disadvantages of GE? How do we over come this?
- ThemaindisadvantageoftheGEisjouleheating.
- Lowervoltage passes through the gel medium,thuslongerseparationtime.
- Overcome:Capillaryelectrophoresis(CE)technique(usingcapillaryinsteadofgelsupportingmedium
briefly describe capillary electrophoresis. What are its main advantages?
•Automated,analyticalversionoftheconventionalelectrophoresistechniques.
•Separationmedium:
Gelmediumreplacedwithfusedsilica capillarytube(10‐100µminternaldiameter,1mlong)filled withbuffer
Main advantages:
High efficiency and easy automation (increases reliability of results)
DCvoltagesourcedeliveringupto30kV
Fastseparationswithverylittlebandbroadening
Samplesize:can be small but detection sensitivity is still good.
what are the steps involved in CE?
(1)Purgecapillarywithelectrolytetoremoveprevioussamplematrixfor1minute
(2)Movecapillaryintonewsampleandallowhydrostaticsampleintroductionfor30seconds.
(3)Placecapillarybackintoelectrolyteandapplyvoltageforanalysistime.Thiscreatesanelectricfieldstrengthacrosscapillary
(4)Ionsseparateincapillaryastheymigratetowardsthedetector
(5)Separatedionspassthroughcapillary’sUVcellwindowandaredetectedaschangesinUVabsorbance.
(6)Datasystemproducesanelectropherogramanalogoustochromatogram
(7)Processelectropherogramusingcomputer.AreaofPeakwidthandreferencepeakforidentificationareused
how do the results of CE appear?
similartoachromatograminchromatographicseparation migration time is recorded. CE can perform both quantitative and qualitative analysis precisely
Why is dna typing performed?
–detectsnormalvariationsinasampleofDNA.
Toestablishidentity,parentage,familyrelationship&appropriatematchesfortransplantationoforgans&tissues
what is restriction fragment length polymorphisms?
–1st techniqueusedforforensicDNAtyping.
DNAcutbyrestrictionenzymes;eachpersonwillhavedifferentDNAfragments
SeparateDNAfragmentsbygelelectrophoresis.
what are the steps involved in RFLP?
A.GenomicDNAdigestionbyrestrictionenzymes
DNAistreatedwithrestriction*endonuclease(enzyme)tocuttheDNAatspecificpoints,resultedindifferentsizedofDNAfragments.
B1.Electrophoresis
DNAfragmentsareseparatedaccordingtosize.
Withphosphategroups,thusDNAfragmentsarenegativelychargedatanalkalinepH.
B2.Denaturation
•SoakedinNaOH&NaClsolution
•DoublestrandedDNAintosinglestrandedDNA
C1.SouthernBlotTechnique
•SinglestrandedDNAistransferredfromthegeltoanylonmembrane.
•DNAprobeisaddedtotheDNAthatisaffixedtothemembrane.
C2.ProbeHybridization
•TheprobewillattachtoasectionofDNA(complementarybasesequence).
•DNAprobeisradioactive&emitsparticles.
•ProbedoesnotbondwiththeDNAiswashedaway
C3.VisualizationbyradioactiveDNAprobe
•Autoradiograph:X‐ray(photo)film.
•EachDNAsampleproducesanimageasbandslocatedinspecificpositions;Possibletocomparetwoormoreautoradiographstoseeifthebandsmatch.
•RFLPislesscommon
‐ItrequireslargeamountsofDNA&canbeeasilycontaminated.
Describe how DNA typing can help to solve cold cases now
DNA databases have been made in many countries which contain DNA information of known criminals
If biological sample of old cold cases are kept in proper condition, we can perform DNA typing and compare against DNA databases. If culprit has criminal record, a match will be found
