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,useofrestrictionenzymes,mappingofplasmidsafterdigestbyrestrictiondigest,gel electrophoresis,amplificiation ofnucleicacidsequences byPCR,mappingofpolymorphismsin“studentpopulation”(Alu repeats,diseaserelatedSNPs),analysisandinterpretationofresults instudentpopulation.

Organization Course: Laboratorio Biologia Molecolare

Theoretical Lections:Prof.Schoeftner(Technologies):04.10.2021– 20.01.2021MONDAY:08:15– 10:15THURSDAY:08:15– 09:15

Laboratory exercises: Prof.ssaBandiera;TurnoI– VI+1experienced supervisor+2tutors16students perturnoinlab;socialdistancing,mascherina

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

Prof.ssa Bandiera:CONTENTS LABORATORY COURSE

Inscription inturniviaMoodlefederato– ASAP!!…this week

Turni:6:16Students/Turno=96Students

Backgroundlectures (Prof.ssaBandiera):

- Monday 04.10.2021- Monday 11.10.2021- Monday 25.10.2021- Monday 15.11.2021- Monday 29.11.2021

Turno1 Turno2 Turno3 Turno4 Turno5 Turno6Genomic DNA preparation 13-Oct 14-Oct 15-Oct 20-Oct 21-Oct 22-OctRestriction digest and gel electrophoresis 27-Oct 28-Oct 29-Oct 10-Nov 11-Nov 12-NovPCR set-up 17-Nov 18-Nov 19-Nov 24-Nov 25-Nov 26-NovPCR analysos 01-Dec 02-Dec 03-Dec 15-Dec 16-Dec 17-Dec

TurniLaboratorio(Prof.ssaBandiera):

DNA polymorphism

GOAL:ApplicationofmolecularbiologytechniquesforthediagnosisandmonitoringofspecificDNApolymorphisminstudentsofthecourse.FocusonAlu repeats

Definition: Polymorphism involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair. Polymorphisms can also be much larger in size and involve long stretches of DNA.

Determination of presence or absence of Alu insert within the PV92 locus in student DNA

Alu repeats inhumans

Karyotype from a female human lymphocyte (46, XX). Chromosomes were hybridized with a fluorescence in situ hybridization probe for Alu elements(green).DNA is counterstainedwith TOPRO-3 (red).Alu elementswereused as a marker for chromosomes and chromosomebands rich in genes.

Throughoutevolution,intron sequences have been thetargetofrandominsertions byshortrepetitive interspersed elements,alsoknownas SINEs.SINEshave become randomly inserted within our intronsovermillions ofyears.One such repetitive element iscalled theAlu sequence

This is aDNAsequence about 300basepairs longthat is repeated,one copyat atime,almost 500,000timeswithin thehumangenome.

Theorigin andfunctionofsuch randomly repeated sequences is not yet known.TheAlu name comes fromtheAlu Irestrictionenzyme recognition sitethat is found inthis sequence.

• 306basepair segmentofDNA,ClassifiedasaSINE (ShortInterspersedRepetitiveElement)• NamedfortheAlu I restrictionsitewithinthesequence(AGCT)• Human-specificAlu insertion• Approx.1millionAlu copiesperhaploidgenome=11%ofthegenome:roleingeneticarchitecture

andgeneticdisorders• Intron:Foundinanon-codingregionofyourDNA

GGCCGGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCGGGCGGATCACGAGGTCAGGAGATCGAGACCATCCCGGCTAAAACGCTGAAACCTCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTAGTGGCGGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCCGAGATCCTGCCACTGCACTCCAGCGTGGGCGACAGAGCGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAAAAAA

306basepairslong:Thissequenceremainsthesame,nomatterwhereitisfoundinthegenome

Transposable elementsinthegenome

Alu polymorphisms canbelinked todisease

• Eachgenelocushasaparticularformofthegene,orallele

• WhatarethepossibleallelesfortheAlu insertateachlocus?+,Alu present-,Alu notpresent

• WhatarethepossiblegenotypesfortheAlu insertforanygivenperson?Homozygouspositive:+/+Homozygousnegative:-/-Heterozygous:+/-

Chromosome 16 Homologous Chromosomes

PV92 Locus

m p

DetectionofAlu polymorphisms instudentpopulation AA2021-2022

- Alu sequenceinsertion inPV92locusisnotdiagnosticforanydiseaseordisorder!- Usefulforforensictests

641bp

941bp

PV92withoutAlu

PV92withAlu

Regiontobeamplified

Regiontobeamplified

Options:

+/+=941/941bp+/- =941/641bp-/- =941/641bp

Transposable elementsinthegenome

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 investigatethegenotypic frequencies fortheAlu polymorphism intheir class populationandcomparethemwiththegenotypic frequencies ofother populations.

COURSEWORK:NODISEASECORRELATION

Contents of Practical Course (Prof. Bandiera)

Applicationofmolecularbiologytechniquesforthediagnosisandmonitoringofspecificgeneticconditions(allelicvariants)andgeneticvariationofAlu repeatinstudentsofthecourse.

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

2. PLASMIDS:Plasmidswillbesubjectedtocontroldigestandfragmentswillbeanalyzedbygel-electrophoresis

3. PREPARATIONOFGENOMICDNA;AnonymizedpreparationofgenomicDNAfromcheekcellsofstudentsanddeterminationofconcentration.

4. PCRAMPLIFICATIONOFSITEOFGENETICALUREPEATVARIANT:Alu repeatsnumbervariationonalocusofchromosome16willbedeterminedbyspecificPCR.AgaroseGelelectrophoresiswillbeusedtomonitordifferencesinAlu repeatnumber.

5. DATAANALYSISANDDISCUSSION:PCRresultswillbeanalyzed;discussiononimprovingPCR;Chi-squareanalysiswillbeusedtotocomparetheAlu genotypefrequencieswithintheclasspopulation.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

Guidelines MSTeamsAA2021-2022

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