presents

(Basic)LaboratoryTechniques (inMolecularBiology)
AMontagud ENavarro PFernndezdeCrdoba JFUrchuegua

DNAcloning
cutandpasteDNA bacterialtransformation
transfection chromosomeintegration

readingandwritingDNA
DNAsequencing DNAsynthesis

molecularhybridization
Southernblot Northernblot Westernblot

cellularscreening cellularculture extractionofDNA

PolymeraseChainReaction
DNApolymeraseDNAdependent PCRdynamics PCRtypes

rewritingDNA:mutations
randommutagenesis pointmutation chromosomemutation

Gelelectrophoresis

arrays

DNAarray protein array LaboratoryTechniques

DNAcloning

DNAcloningoverview

LaboratoryTechniques

cutandpaste DNA
jointwoDNAmolecules
insert:usuallysmaller vector:hasoriginofreplication

Figure830. The insertion of a DNAfragment into abacterial plasmid with the enzyme DNA ligase.(Alberts etal, 2002)

LaboratoryTechniques

cutandpaste DNA: vectors

bacteria:plasmids
restrictionsites resistancegene originofreplication
Figure3.22.(Cooper,2000)

eukaryote:viruses
restrictionsites virusgenes terminalrepeats

LaboratoryTechniques

cutandpaste DNA: restriction

restrictionnucleasesenzymescutDNA
fromunspecific(exonucleases)tohighlyspecific (typeIIendonucleases) leavesbluntorstickyends

rotational symmetry!!

Figure821.(Alberts etal,2002)

(Alberts LaboratoryTechniques

Figure822. etal,2002)

cutandpaste DNA: restriction

whystickyends?

Figure821.(Alberts etal,2002)

Figure77. Ligation of restriction fragments with complementary LaboratoryTechniques sticky ends.(Lodish etal,2000)

cutandpaste DNA: ligation

DNAligase

Figure514.The reaction catalyzed byDNAligase.(Alberts etal,2002)

Figure77. Ligation of restriction fragments with complementary LaboratoryTechniques sticky ends.(Lodish etal,2000)

bacterialtransformation
makecellmembranepermeable introduceplasmidDNAintobacteria manydifferentprotocols

Figure716. Bacterium undergoing transformation.(Grifftihs etal,2000)

LaboratoryTechniques

transfection
useofvirusestogetDNAintocells ineukaryoteandbacteria(transduction)

Figure726.The mechanism of generalized transduction. (Griffithsetal,2000)

LaboratoryTechniques

chromosomeintegration
howtomakestablemutants addsagenetoa chromosome homologousrecombination
targetDNA

possibleinbacteria usuallynecessaryin eukaryote

Figure575.Transpositional sitespecific recombination byaretrovirusor aretrovirallike retrotransposon.(Alberts etal,2002) LaboratoryTechniques

cellular screening

Figure126. Two plasmids designed asvectors for DNAcloning,showing generalstructure and restriction sites.(Griffithsetal,2000)

LaboratoryTechniques

cellularculture
getlotsofcells solidorliquidmedia timevaries
E.coli:overnight humancells:days

adapted from Alberts etal,2002 LaboratoryTechniques

extractionofDNA
takeonlyDNAfromthewholecellextract
brakecellmembrane precipitateproteins

purifyDNAfromproteinsbound,etc

LaboratoryTechniques

extractionofplasmids
plasmids
smallcircular independentDNA molecules

extractonly plasmids takeadvantage

smaller,morecompact

a.k.a.maxi/midi/mini prep
Figure122.(Griffithsetal,2000) LaboratoryTechniques

DNAcloningoverview,again
restriction+ ligation plasmidextraction

transformation cellular screening cell culture

LaboratoryTechniques

movie :DNAcloning

Ch7anim1.Lodish etal,2000

LaboratoryTechniques

PolymeraseChainReaction: copyingDNA

DNApolymeraseDNAdependent
copiesDNAintoDNA DNAreplication addsdNTP toa3OHendofanexistingstrand

LaboratoryTechniques

Figure54. DNAsynthesis catalyzed byDNApolymerase. (Alberts etal,2002)

PolymeraseChainReaction
doublestrandedDNA primers heattolerantDNApolymerase(Taq pol) dNTPs
95C 55C 72C Figure839.Amplification of DNAusing the PCRtechnique (Alberts etal,2002)

LaboratoryTechniques

PCRdynamics

95C

72C 55C

LaboratoryTechniques

PolymeraseChainReaction

Figure839.Amplification of DNAusing the PCRtechnique.(Alberts etal,2002) LaboratoryTechniques

PCRamplifiesDNAcopies

LaboratoryTechniques

movie :PCR

Ch7anim4.Lodish etal,2000

LaboratoryTechniques

PCRtypes
QPCR AllelespecificPCR AssemblyPCR ColonyPCR InversePCR LigationmediatedPCR NestedPCR

andmanymore...

LaboratoryTechniques

Gelelectrophoresis: separatingmoleculesperlength

Gelelectrophoresis
nucleicacidsinan agarose gel electriccurrentthrough thegel nucleicacids
DNAorRNA migratetothe+ pole
Pskeletonhas charge

separatedperlength dyed(EtBr,SYBRgreen)

Figure722. Separation of DNAfragments of different lengths bygelelectrophoresis. LaboratoryTechniques (Lodish etal,2000)

Gelelectrophoresis
usuallythelengthis inferredusingaknown sample proteins
SDSPAGEgel canbe2D:pI andweight

+
an EtBr gelelectrophoresis Figure817. (Alberts etal,2002)
LaboratoryTechniques

readingandwritingDNA

DNAsequencing
allowstoknowwhatistheexactsequence ofA,T,C,GofaDNAmolecule Sangermethod(1977)
basedonPCRreaction&gelelectrophoresis ddNTPs radioactivelylabelled

LaboratoryTechniques

DNAsequencing

Figure836. The enzymatic or dideoxy method of sequencing DNA. (Alberts etal,2002)

LaboratoryTechniques

movie :Sanger sequencing

Ch7anim3.Lodish etal,2000
LaboratoryTechniques

DNAsequencing: present&future
fluorescencelabels capillar electrophoresis polonies nanopores pyrosequencing
LaboratoryTechniques

DNAsynthesis
commercialsynthesis
currentprice:from0,80/bp productiontime:from2weeks

from Carlson,2003
LaboratoryTechniques

molecularhybridization

molecularhybridization
nucleicacidsspecificallyhybridizeto nucleicacids usinglabelledn.a.,specificdetection ispossible

Figure557. DNAhybridization.(Alberts etal,2002)

Figure717. Membranehybridization assay for detecting nucleic acids. LaboratoryTechniques (Lodish etal,2000)

molecularhybridization

incombinationwithgel electrophoresis, detection boastsitspotential

Figure827.Detection of specific RNAor DNAmolecules LaboratoryTechniques bygeltransferhybridization.(Alberts etal,2002)

molecularhybridization
Southernblot(DNA)
DNAextraction restriction gelelectrophoresis denaturation filtertransfer labelledprobehybridisation
DNAorRNA

Northernblot(RNA)
RNAextraction denaturation gelelectrophoresis filtertransfer labelledprobehybridisation
DNA

detection

detection

Westernblot(protein)
polyacrilamide gelseparation filtertransfer probereaction
antibody

LaboratoryTechniques

detection

Southern,Northern,Western

Figure1020.Comparison of Southern,Northern,and Westernanalyses of GeneX. (Griffithsetal,2000)

LaboratoryTechniques

rewritingDNA: mutations

mutations
variationsonagivenDNA molecule basisofvariability evolution smallscaletypes
silentmutation:a.a.isnotafected missense mutation:differenta.a. nonsensemutation:a.a. stop

Figure84.Different types of mutations. (Lodish etal,2000)

LaboratoryTechniques

mutations
largescaletypes: chromosomes
inversion:changesorder insertion:addsgenes deletion translocation:movesgenes

Figure84.Different types of mutations. (Lodish etal,2000)

LaboratoryTechniques

randommutagenesis
usechemicals,UVorerrorproneDNA replication finescreeningneeded!

Figure162.Mismatched bases. (Griffithsetal,2000)

Figure549. Howchemical modifications of LaboratoryTechniques nucleotides producemutations.(Alberts etal,2002)

pointmutation
pointmutationona givensite PCRiswidelyusedfor thisgoal
sitedirected megaprimers invitro overlap extension
Figure869.The useof asynthetic oligonucleotide to modify the proteincoding region of ageneby LaboratoryTechniques sitedirected mutagenesis.(Alberts etal,2002)

movie :PCRmutagenesis

Ch8anim1.Lodish etal,2000
LaboratoryTechniques

chromosomemutation
mutate chromosomes
knockout replace insertion:addagene deletion

homologous recombination
targetDNA

Figure864.Genereplacement, geneknockout,and geneaddition. (Alberts etal,2002)

LaboratoryTechniques

chromosomemutation

Figure873.Making collections of mutant organisms. (Alberts etal,2002)

LaboratoryTechniques

arrays

DNAarray
probe
DNAmolecules of variablelenght on asolid support inaregularand fixed distribution

sample
labelled DNAor RNA that will bind to the probes
Figure1427.(Griffithsetal,2000)

takeadvantageofnucleic acidsspecifichybridization

LaboratoryTechniques

expressionarray
probe
usually organisms ORFs ordered (oligo chip)

sample
usually labelled mRNA or retrotranscribed mRNA (cDNA)

LaboratoryTechniques

expressionarray
monitormRNA quantitiesofthewhole genome comparetwostates
inputs
sample 1 sample 2

outputs
sample 1>sample 2 sample 1<sample 2 sample 1=sample 2

Figure862.Using DNAmicroarrays to monitorthe expression of LaboratoryTechniques thousands of genessimultaneously.(Alberts etal,2002)

expressionarray:breastcancer
84 number of experiments 1753 genes

below median median

LaboratoryTechniques

above median Perou etal (2000)

expressionarray:breastcancer

Perou etal (2000)

LaboratoryTechniques

protein array
probe
protein molecules on a solid support inaregular and fixed distribution

sample
substrates that will bind to the probes proteins that could interact with probes takeadvantageofprotein antibodies that could substraterecognition recognize the probes LaboratoryTechniques

sources
Alberts etal,MolecularBiologyoftheCell,GarlandScience,4thed,2002 Lodish etal,MolecularCellBiology,Freeman&Co.,4thed.,2000 Cooperetal,The Cell AMolecularApproach,Sinauer Publishers,2nd ed.,2000 Griffithsetal,AnIntroductiontoGeneticAnalysis,Freeman&Co.2000 Carlson.Thepaceandproliferationofbiologicaltechnologies. Biosecurity andBioterrorism.2003 Perou etal,Molecularportraits of humanbreast tumours.Nature. 2000 www.ergito.com

LaboratoryTechniques

farewell movie :the PCRsong

http://www.youtube.com/watch?v=x5yPkxCLads YouTube
LaboratoryTechniques

the PCRsong
There was atimewhen to amplify DNA, you had to grow tons and tons of tiny cells. Then along came aguy named Dr.Kary Mullis, said you canamplify invitro just aswell. Just mixyour template with abufferand some primers, nucleotides and polymerases,too. Denaturing,annealing,and extending. Well its amazing what heating and cooling and heating will do. PCR,when you need to detect mutations. PCR,when you need to recombine. PCR,when you need to find outwho the daddy is. PCR,when you need to solve acrime.
LaboratoryTechniques

more!
BiotechNation Feat.Notorious GFP Transformation BiotechNation Restriction enzymes

http://www.youtube.com/watch?v http://www.youtube.com/watch?v =Gy21KhSF3PM =cFeNlM1gJoo YouTube YouTube LaboratoryTechniques