Topic 3 Biochemical Analytical Technique Part-2

Question 1

what is dna?

Answer 1

Deoxyribonucleicacidcontain genetic material in its nucleus and contains 23 pairs of chr

Question 2

what are the monomeric units of DNA and RNA respectively?

Answer 2

deoxyribonucleotidesand

ribonucleotides.

Question 3

what are the 3 components of DNA?

Answer 3

a)Nitrogenousheterocyclicbase
(purineorpyrimidine)

b) Pentosesugar
c) Phosphategroup

Question 4

what are the 4 types of bases in DNA?

Answer 4

Adenine,Thymine,
Cytosine, Guanine
AdeninelinkswithThymine CytosinelinkswithGuanine

Question 5

what are the 2 diff between dna and rna

Answer 5

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

Question 6

what is a protein?

Answer 6

Madeofaminoacids
Containbothacidiccarboxyl(‐COOH)and
basicamino(‐NH2)
groupattachedtoαcarbonatom.
DifferwithrespecttoRgroup.

Question 7

how is a protein formed?

Answer 7

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.

Question 8

what is a Zwitterionic aminoacids?

Answer 8

Whendissolveinwater
theycontainanaminegroup(basic)andacarboxylicgroup(acidic)
Thenetchargeoftheentiremoleculeiszero.

Question 9

how to calculate isoelectric pH?

Answer 9

IsoelectricpH=(pK1 +pK2 )/2

Question 10

what is the isoelectronic pH?

Answer 10

MoleculehasnonetelectricchargeatthispHandwillnotmoveinanelectricfield

aminoacidhastheleastbufferingeffect (no ability to change the pH of a system)

Question 11

what are some examples where DNA can be collected?

Answer 11

Couldbealickedenvelope,dirtylaundry,acigarettebuttorsalivaetc.

Question 12

what are some Specialprecautionswe should take whenhandlingspecimens:

Answer 12

gloves,disposableinstruments,avoidtalkingandsneezing,avoidtouchingsamplewithyourskin,air‐drytheevidencebeforepackagingsomolddoesnotgrow

Question 13

what degrades evidence?

Answer 13

sunlight,hightemperatures,bacteria,

moisture

Question 14

what is the ideal sample?

Answer 14

e:1mLoffresh,wholeblood(whitebloodcells)treatedwithEDTA

**Ethylenediaminetetraaceticacid(EDTA)stronglyandirreversiblychelates(binds)calciumions,preventingbloodfromclotting

Question 15

what are the 3 bioanalytical techniques for DNA typing?

Answer 15

PolymeraseChainReaction(PCR)
RestrictionFragmentLengthPolymorphisms(RFLP)
electrophoresis

Question 16

what is the function for PCR?

Answer 16

ToamplifyminutequantitiesofDNAespeciallywhenmoreDNAtemplateisrequiredforfurtherreactions/analysis

Question 17

what is the function for RFLP?

Answer 17

TofragmentDNAsamplesintopieces,utilizeshomologousDNAdifferenceswithinrestrictionenzymebindingsites.

Question 18

what is the function for electrophoresis?

Answer 18

Toseparatethefragmentsbasedonchargedandlength/size.

Question 19

what is the PCR test mainly for? what is its main advantage?

Answer 19

‐Techniqueformakingcopiesoramplifyinga
specificsequenceofDNAinashortperiodoftime.

‐CellfreeDNAamplification

ConvenientmethodforobtaininglargenumberofDNAcopies.Canbethoughtofasamolecularphotocopier.

advantage:
RobustandworksforsmallamountsofDNAandworkseveniftheDNAisbadlydegraded.

Question 20

what are the basic steps of PCR? (DAE)

Answer 20

1. •Denaturation:
   –DNAfragmentsareheatedathightemperatures(94oC)–DNAdoublehelixreducedtosinglestrands.
2. •Annealing:
   –Thereactionmixtureiscooleddown.
   –PrimersannealtothecomplementaryregionsintheDNAtemplatestrands
   –Doublestrandsareformedagain.
3. Extension: TheDNApolymerasesynthesizesthecompletecomplementarystrand.

Question 21

how do we calculate the number of DNA produced in a PCR cycle?

Answer 21

No of DNA produced = 2^n

n = no of cycles

Question 22

what is electrophoresis?

Answer 22

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.

Question 23

what is the rate of migration of a given species dependent on?

Answer 23

dependsuponcharge and sizeofions.

Question 24

what is the unique ability of electrophoresis? what is it commonly used for?

Answer 24

•Uniqueabilitytoseparatechargedmacromolecules ofinterest. •PopularforanalysisofDNA,RNAandprotein

Question 25

what are the 3 principles of separation of DNA fragments in electrophoresis?

Answer 25

Formofseparationusedtoidentifymacromoleculesofbiologicalcompoundse.g. DNAandproteins

Movementofchargedparticlesinanelectricfield.Anegativelychargedparticlewillmovetowardthepositiveendandviceversa.ProteinsarechargedatallpHexceptattheirisoelectricpoint(pI).

Differencesinsizeandchargebetweenproteinsresultsindifferentmigrationrateswhentheyareinanelectricfield.

Question 26

what are the 2 apparatus used in electrophoresis?

Answer 26

(a) D.C.powersupplywithcontrolforbothvoltageandcurrentoutput.
(b) Electrophoresisunitcontainingelectrodes,buffersreservoirandasolidsupportforelectrophoresismedium.

Question 27

explain the general principles of electrophoresis technique in terms of zwitterions, cations and anions

Answer 27

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

Question 28

what are the 2 types of electrophoresis?

Answer 28

a) Gelelectrophoresis(Supportingmedium:Gel):
(i) Starch
(ii) Agarose(Agarosegelelectrophoresis)
(iii) Polyacrylamide(SodiumDodecylSulphate–PolyAcrylamide Gel Electrophoresis)

b)Capillaryelectrophoresis(Supportingmedium:Capillary)

Question 29

briefly describe starch as a supporting medium for electrophoresis. what is its application, advantage and disadvantage?

Answer 29

Starchassupportingmedium: Preparebyheatingandcoolingmixtureofpartiallyhydrolyzed starchinanappropriatebuffer.

Disadvantage:

(1) Nowaytodetermineexactporesizeofstarchgels.
(2) Differentbatchesareinconsistentwithrespecttoporesizeforsamepercentageofstarch.

Advantage: Easeofapplyinghistochemicaltests.

Application: Analysisofisoenzyme patterns.

Question 30

briefly describe agarose gel as a supporting medium for electrophoresis.

Answer 30

SeparationofDNAfragments>1000basepairs.
AllDNAisofuniformshapeandcharge‐massratio.
Separatedbymolecularweight/sizeandmeasuredasbasepairs.

Question 31

what are the steps involved in agarose gel electrophoresis?

Answer 31

•Sampleisappliedasaspotonawellinsupportingmediumofelectrophoresiskit.

•ApplyD.C.andtheionsofthesamplemigratetowardstheelectrodesofoppositepolarityatcharacteristicratestoformbands

•Migrationrateisdependentonchargeandoncompletionofelectrophoretogram,thesupportingmediumisdriedandsamplecomponentsaredetected

•Proteinmoleculescanbeseparatedfromeachotherbytakingadvantageoftheiroverallcharged(positiveornegative).

•Inelectricfieldbetweentwoelectrodes,apositivelychargedparticlewillmovetowardnegativeelectrodeandanegativelychargedparticlewillmovetowardpositiveelectrode.

Question 32

briefly describe polyacrylamide gel as a supporting medium for electrophoresis.

Answer 32

Cross‐linkedpolymer:
suppressconvectivecurrentsproducedbysmalltemperature
gradients,arequirementforeffectiveseparation.

•serveasmolecularsievesthatenhanceseparation.

Question 33

why is polyacrylamide a commonly used gel medium?

Answer 33

(a) chemicallyinert
(b) readilyformedbypolymerisationofacrylamide.
(c) Poresizecontrolledbychoosingvariousconcentrationsofacrylamide(0.7–2%).
(d) highdegreeofreproducibility&resolution

Question 34

describe how the qualitative results of gel electrophoresis comes about

Answer 34

Mobilityofproteinsislinearlyproportionaltothelogarithmoftheirmass.

Question 35

what is the main disadvantages of GE? How do we over come this?

Answer 35

• ThemaindisadvantageoftheGEisjouleheating.
• Lowervoltage passes through the gel medium,thuslongerseparationtime.
• Overcome:Capillaryelectrophoresis(CE)technique(usingcapillaryinsteadofgelsupportingmedium

Question 36

briefly describe capillary electrophoresis. What are its main advantages?

Answer 36

•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.

Question 37

what are the steps involved in CE?

Answer 37

(1)Purgecapillarywithelectrolytetoremoveprevioussamplematrixfor1minute
(2)Movecapillaryintonewsampleandallowhydrostaticsampleintroductionfor30seconds.
(3)Placecapillarybackintoelectrolyteandapplyvoltageforanalysistime.Thiscreatesanelectricfieldstrengthacrosscapillary
(4)Ionsseparateincapillaryastheymigratetowardsthedetector
(5)Separatedionspassthroughcapillary’sUVcellwindowandaredetectedaschangesinUVabsorbance.
(6)Datasystemproducesanelectropherogramanalogoustochromatogram
(7)Processelectropherogramusingcomputer.AreaofPeakwidthandreferencepeakforidentificationareused

Question 38

how do the results of CE appear?

Answer 38

similartoachromatograminchromatographicseparation migration time is recorded. CE can perform both quantitative and qualitative analysis precisely

Question 39

Why is dna typing performed?

Answer 39

–detectsnormalvariationsinasampleofDNA.

Toestablishidentity,parentage,familyrelationship&appropriatematchesfortransplantationoforgans&tissues

Question 40

what is restriction fragment length polymorphisms?

Answer 40

–1st techniqueusedforforensicDNAtyping.

DNAcutbyrestrictionenzymes;eachpersonwillhavedifferentDNAfragments

SeparateDNAfragmentsbygelelectrophoresis.

Question 41

what are the steps involved in RFLP?

Answer 41

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.

Question 42

Describe how DNA typing can help to solve cold cases now

Answer 42

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