Prof. SCHOEFTNER STEFAN, responsible corso – Lecture

Prof.ssa BANDIERA ANTONELLA – Laboratory course

Insegnamento:LaboratorioBiologiaMolecolare

Docenti:

The course provides theoretical and practical training on techniques and experimental approaches in molecular biology.

- Afocuswillbesetonthemolecularbiologyandtechnologies relatedtoofnucleicacids

- BasictechniquesforDNAmanipulation,genestudy,genecloning,geneexpressionanalysisandrecombinantDNAtechnologywillbeaddressed;genomebrowsersearch.

- Laboratoryexercises includetheteachingoflaboratorysafetystandardsthehandlingoflaboratoryinstruments,theextractionofDNAfrombacteriaandhumancheak cells,useofrestirction enzymes,mappingofplasmidsafterdigestbyrestrictiondigest,gel electrophoresis,amplificiation ofnucleicacidsequences byPCR,mappingofpolymorphismsin“studentpopulation”(Alu repeats,diseaserelatedSNPs),analysisandinterpretationofresults instudentpolylation andoverallpopulation.

Organization Course: Laboratorio Biologia Molecolare

Monday 30.09.2019– 28.10.2019:11hourstheroretical lections:Prof.Schoeftner(Technologies)

Monday 04.11.2019– 16.12.2019:Theroretical lections:Prof.Schoeftner(Technologies)Laboratory course:Prof.Scaggiante

Monday Lecture:ca.1,5hours:Theoretical lecture (Schoeftner)– related totopic inweeklylaboratory exerciseca.0,5hours:Prof.Scaggiante – practical backgroundtolaboratory background

Labortory exercises:TurnoI– IV;all “turniwill dothesame porgram intherespective weekProf.Bandiera+2experienced tutors(20-25stundents perturno;4-5students perworkstation

7.Exercisesession: StudentsgetintroductionintotheuseoftheENSEMBLgenomebrowserandprimerconstruction.Studentswilldoprimerdesignforpracticalpartashomework.

Contents of Theoretical Lecture (Prof. Schoeftner)

1.Anatomyofthecell,biomolecules, conceptofpreparationofRNA/Protein/DNA.

2.RecombinantDNAtechniques,Cloningvectors,endonucleases, artificialchromosomes,recombinantproteinexpression,introductionofgenesintohost-organisms.

3.DNAsequencing,bacterial immunity,manipulationofthegenomecontentofpro- andeukaryoticorganisms,siRNA/shRNA mediatedknock-downapproaches.

4.Hybridizationrelatedtechniques(RNA-FISH,DNA-FISH,Southernblot,Northernblot),Electrophoresis,methodstostudyDNA:protein interaction(bandshift,DNAfootprinting,chromatinimmunoprecipitation)

5.PCRtechnologies: standardPCR,RT—PCRandvariants

6.Geneexpressionanalysis:arraytechnologyandhighcontentsequencing, determinationof3’and5’endsofRNA,singlemolecule transcriptanalysis

Contents of Practical Course (Prof. Bandiera)Applicationofmolecularbiologytechniquesforthediagnosisandmonitoringofspecificgeneticconditions(allelicvariants)andgeneticvariationofAlu repeatinstudentsofthecourse.

1. THEMOLECULARBIOLOGYLABORATORY:Ruleofconductandsafety,hazardousreagentsandmaterialsafetydatasheet;equipmentandlabinstrumentation.Theuseofautomaticlabpipettesforsmallvolumemanipulation.

2. PLASMIDS:PlasmidDNAextractionbyacommercialkit,evaluationofextractionyield,preparationofsamplesforelectrophoreticanalysis.Plasmidswillbesubjectedtocontroldigestusingrestrictionenzymesandwillserveaspositiveandnegativecontrol forsubsequentPCR.

3. PREPARATIONOFGENOMICDNA:4. AnonymizedpreparationofgenomicDNAfromcheekcellsofstudentsanddeterminationofconcentration.

5. PCRAMPLIFICATIONOFSITEOFGENETICALUREPEATVARIANT:Alu repeatsnumbervariationonalocusofchromosome16willbedeterminedbyspecificPCR.AgaroseGelelectrophoresiswillbeusedtomonitordifferencesinAlu repeatnumber.

6. PCRAMPLIFICATIONOFSITEOFALLELICG6PDVARIANTS:Alocusharboringallelic variantsoftheG6PDgenewillbeamplifiedbyPCR.PCRproductswillbepurifiedaftergelelectrophoresisandsubjectedtodigestusingrestrictionenzymes.DigestedDNAwillbeseparatedbygelelectrophoresis.

8.DATAANALYSISANDDISCUSSION:Chi-squareanalysiswillbeusedtotocomparetheAlu genotypefrequencieswithintheclasspopulationwiththosepredictedbytheHardy-Weinbergequation.Thegenotypicfrequenciesoftheclasspopulationcanalsobecomparedwiththegenotypicfrequenciesofanotherpopulationinthedatabase.

à 2writtenexams:

Exam1:Reportsonlabworkattheendofeachlabpractice(Prof.Bandiera).Reportswillbeevaluatedassessing:-diligence,attendance,presentationaccuracy-personalskills,synthesis,descriptionandclarityinpresentation,technicaltermsknowledge-understandingdegree,explanationanddiscussion skills,presenceofconceptualerrors.à Atotalof15pointscanbereached.àAminimumof7,5pointsisnecessarytoparticipateinthesecondpartoftheexam2

Exam2Learningprogressonthetheoretical lectures(Prof.Schoeftner)willbemonitoredinawrittenexam.Totalpoints:16.Exam2consistsof12multiple choicequestions(0,5pointsperquestion)and2“openquestions”(5pointsperquestion,max1pageanswertoquestion)onbroadertopicsaddressedduringthetheoretical lecturesandvirtuallab.

Thefinalmarkofthecourseresultsfromthesumofbothexams.Maximumpoints:31A minimumof18pointsisrequiredtopasstheexam“Laboratorio BiologiaMolecolare”.

Exam

Turni:5Inscription inturniviaMoodlefederatoinca.2weeks!

CONTENTS LABORATORY COURSE

1. Preparation of Genomic DNA

2. Determination of presence or absence of Alu insert within the PV92 locus

Alu repeats inhumans

Karyotype from a female human lymphocyte (46, XX). Chromosomes were hybridized with a probe for Alu elements (green) and counterstained with TOPRO(red).Alu elements were used as amarker for chromosomes and chromosomebands rich in genes.

Throughoutevolution,intron sequences have been thetargetofrandominsertions byshortrepetitive interspersed elements,alsoknownas SINEs.7SINEshave become randomly inserted within our introns overmillions ofyears.One such repetitive element iscalled theAlu sequence7(Figure2).This is aDNAsequence about 300basepairs longthat is repeated,one copyat atime,almost500,000timeswithin thehumangenome.8Theorigin andfunction ofsuch randomly repeated sequences is not yet known.TheAlu name comes fromtheAlu Irestriction enzyme recognition sitethat is found inthis sequence.

2. Determination of presence or absence of Alu insert within the PV92 locus

1 2 43 5 6 Student

PCR amplification usingspecifc primers +gel-

electrophoresis

AlleleA

AlleleB

Alleles (pat/mat)A/A B/B B/B A/A B/B A/B

Studentswill perform abioinformatics exercise toinvestigatethegenotypic frequencies fortheAlupolymorphism intheir class population andcomparethemwiththegenotypic frequencies ofotherpopulations.

Alu repeats:Throughoutevolution,intron sequences have been thetargetofrandominsertions byshortrepetitive interspersedelements,also knownas SINEs.7SINEshave become randomly inserted within our introns overmillions ofyears.One suchrepetitive element is called theAlu sequence7(Figure2).This is aDNAsequence about 300basepairs longthat is repeated,onecopyat atime,almost 500,000timeswithin thehumangenome.8Theorigin andfunction ofsuch randomly repeated sequencesis not yet known.TheAlu name comes fromtheAlu Irestriction enzyme recognition sitethat is found inthis sequence.

COURSEWORK:NODISEASECORRELATION

Ex.6

Chr.X

Glucose-6-phosphatedehydrogenase deficiency (G6PDD)is aninborn error ofmetabolism that predisposes tored blood cell breakdown.Most ofthetime,thosewho areaffected havenosymptoms.Following aspecifictrigger,symptoms such as yellowish skin,darkurine,shortness ofbreath,andfeelingtiredmay develop.Complications canincludeanemiaandnewborn jaundice (yellow pigmentation).Somepeople never havesymptoms

It is anX-linked recessivedisorderthat results indefective glucose-6-phosphatedehydrogenaseenzyme.Red blood cellbreakdownmay betriggered byinfections,certainmedication,stress,orfoods such as favabeans.Depending onthespecificmutation theseverity oftheconditionmay vary.Diagnosis isbased onsymptoms andsupported byblood tests andgenetic testing(PCR-RFLP)

3. Determination of a G6PD variant by RFLP

3. Determination of a G6PD variant by RFLP

<1%

Many variants have been determined (>190)

3.DETECTIONOFG6PD563CàTVARIANT

R =sitoperenzimadirestrizioneMboII

563C 563T

MboIIcuts 2x

MboIIcuts 3x

G6PD563CàTVARIANTINEXON6

1. G6PDExon 6specific primers2. PCRamplify specific region of

students3. Purify PCRproduct4. Digestpurified DNAusing MboII5. Run agarose gel6. 563CàTvariants results anew

MboII site inthePCRfragment7. Additional bandappears ingel

Materiale Didattica

1.RECOMBINANTDNATECHNIQUES

• Dimensioni: circa dieci volte piu’ grandi delle cellule procariotiche (10-100 μm)

• La membrana plasmatica racchiude il materiale cellulare, lo separa dall’ambiente e regola il passaggio di sostanze cellula/esterno

• Compartimentazione interna: all’interno della membrana si trova il citoplasma, l’insieme del contenuto cellulare, comprendente il citosol (soluzione acquosa di piccole e grandi molecole) ed una serie di organuli, compartimenti funzionalmente specializzati delimitati da membrana o comunque strutturalmente separati (Apparato di Goghi; Mitocondrio; Reticilo endoplasmatico)

10-100µm

Genoma humano:3,289,000,000 nucelotidi

Lacellula eucariote

Genomaumano aploide:3.2x 109bp(3200000000bp)

à 22autosomià eterocromosomi (Xed Y)à 23000geni

Dimensione dei cromosomi:45-275Mb;à 2.9x 109bp:eucromatina=attivoàGenomanoto:>90%dell’eucromatina.

L’utillizodella infromazione genetica:

5.000-10.000geni espressi daogni cellula» 100.000specieproteiche diversepermodificazioni post-traduzionali» 108 specieproteiche diversenel genere umano (plasma:proteomadi proteomi)

ENORMECOMPLESSITA

Genomes

Mappagenicaumana

Leregioniinrossoindicanoporzionideicromosomiadaltadensitàgenica (adesempio icromosomi15,16,17,19,20e22).

Altricromosomicome4,18,XeYmostranounacolorazionerossamoltodeboleesonopoveridigeni.

Genenumbers indifferent organisms

DNAmitocondriale dell’uomo:16569paiadibasie37geni(codificanoper13polipeptidisintetizzatidalribosomamitocondriale22tRNA e2rRNA),coinvoltinellaproduzionediproteinenecessarie allarespirazionecellulare.

MITOCHONDRIALDNA

Nucleosome:8histoneproteins2giri di DNA(146nt)

Lacromatinaè laformaincuigli acidi nucleici sitrovano nella cellula.Funzione:- impacchettamento delDNA-rafforzare il DNAperpermetterelamitosi- preveniredanni alDNA-controllare lareplicazionedelDNAe l'espressione(attivita)delgene

LACROMATINA

Lecelluleprocariotiche (dapro,primaekaryon,nucleo)sonoprivediunnucleoracchiusodaunamembrana.

Gliorganismiunicellularicostituitidacelluleprocariotiche,iprocarioti, sonoclassificatiinduedomini:

•Archaea (archei);•Bacteria (batteri).

PROCARIOTI

PROCARIOTI- EUCARIOTI

• In aggiunta al DNA principale i batteri possono contenere piccole molecole di DNA circolare, dette plasmidi, che codificano per enzimi catabolici, per la resistenza ad antibiotici o legati a meccanismi per lo scambio di materiale genetico tra organismi.

• Genoma: 130.000 – 14.000.000 nucleotidi

Il materiale genetico, il DNA, e’ organizzato in un singolo cromosoma circolare, localizzato nell’area nucleare o nucleoide, una regione della cellula non delimitata da membrana.

1-2µm(1.000.000µm=1m)

DNA RICOMBINANTEtecnica che permette di

v ottenere brevi segmenti di DNA clonati e di studiarne la sequenza nucleotidica

v di trasferirli nel genoma di altre cellule

v di controllare l’incorporazione e l’espressione del DNA clonato

v di introdurre mutazioni nel DNA e di studiarne gli effetti

AGeneralStrategytostudy oruserecomobinantDNA