enzimi di restrizione
DESCRIPTION
ENZIMI DI RESTRIZIONE. Reazione ligasica di frammenti di restrizione. DNA RICOMBINANTE: DUE MOLECOLE DI DNA VENGONO UNITE IN PROVETTA SFRUTTANDO LE PROPRIETA’ DI ENDONUCLEASI DI RESTRIZIONE DI TIPO II E DELLA DNA LIGASI. CLONAGGIO - PowerPoint PPT PresentationTRANSCRIPT
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ENZIMI DI RESTRIZIONE
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Reazioneligasica di frammenti direstrizione
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DNA RICOMBINANTE: DUE MOLECOLE DI DNA VENGONOUNITE IN PROVETTA SFRUTTANDO LE PROPRIETA’ DI ENDONUCLEASI DI RESTRIZIONE DI TIPO II E DELLA DNALIGASI
CLONAGGIO-IN BIOLOGIA CLONARE UN ORGANISMO SIGNIFICA OTTENERE UN INDIVIDUO UNA POPOLAZIONE DI ORGANISMI CHE SONO GENETICAMENTE IDENTICI
-IN BIOLOGIA MOLECOLARE CLONARE UN TRATTO DI DNA SIGNIFICAOTTENERE UNA POPOLAZIONE DI DNA IDENTICHE ALLA MOLECOLA DI PARTENZA
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Vettori e clonaggio
Vettori
PlasmidiFagiCosmidiYAC
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Vettori e clonaggio
Plasmide
Molecola circolare di DNA a doppiofilamento, normalmente presentenella cellula batterica, in gradodi replicarsi autonomamente
dal cromosoma.
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VETTORI DI CLONAGGIO
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Bromo-chloro-indoyl--galactopyranosidase or X-Gal (Clear)
Bromo-chloro-indoyl (Deep blue insoluble)
+
galactose
-galactosidase
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Bacteria from ligation platedon ampicillin and X-Gal
Containswild typeplasmd
Containsrecombinantplasmd
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Vettori e clonaggio
Lunghezza massima delDNA clonabile in un plasmide:
circa 20 Kb
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Vettori e clonaggio
I virus sono piccoli parassitinon in grado di replicarsi.Dopo aver infettato una
cellula-ospite utilizzano i suoisistemi di replicazione e sintesi
delle proteine per riprodursi
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Vettori e clonaggio
I batteriofagi (o fagi) sono virus che infettano i batteri.
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Vettori e clonaggio
Il fago più utilizzato in biologiamolecolare è il fago
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Vettori e clonaggio
Assemblaggiodi un fago
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Vettori e clonaggio
Mappa del genoma del fago
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Vettori e clonaggio
Clonaggio diframmenti diDNA nelfago
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Vettori e clonaggio
Formazione di cloni fagici
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Vettori e clonaggio
L’infezione dei batteri con il fago è circa 103 volte più efficiente
della trasformazione con un plasmide
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Vettori e clonaggio
Lunghezza del DNA
clonabile in un fago:
20-25 Kb
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Vettori e clonaggio
Un cosmide è un plasmidecontenente la sequenza COS
dal fago
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Vettori e clonaggio
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Vettori e clonaggio
Clonaggio diframmenti diDNA in uncosmide
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Vettori e clonaggio
Lunghezza del DNA
clonabile in un cosmide:
circa 45 Kb
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EcoR I
Genomic LibraryInfect cells
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AAAAAA AAAAAA
AAAAAAAAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAA
AAAAAAAAAAAA
AAAAAA
AAAAAA
AAAAAA
cDNA synthesis
Infect cellscDNA library
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• Preparing the insert– Partial digestion preferred
Genomic Library Construction
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Genomic library cDNA library
Genomic DNA mRNASource
Species or strains Species or strainsTissuesDevelopmental stages
Variation
12k -- 20k 0.2k -- 6kInsert size
Equal Correlate with expression level
Representation
Only one Expression vs. nonexpression
Type
DNA DNA or antibody orprotein
Probe
Gene structureInfer protein identity
Encoded proteinInfer protein identity
Purpose
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Screening with DNA probe
• Probe is identified cloned– From other organism
– Same organism• Looking for variants
• Chromosome walk
Chromosome walk
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Genome Projects
• Whole genome sequencing
• Fragment and clone• Sequence ALL clones!• Computer assembles
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Genome Projects
Bee Cat Chicken Cow Dog Fruit fly Human Malaria parasite Microbial Genomes Mosquito Mouse Nematode Pig Plant Genomes Central Rat Retroviruses Sea urchin Tribolium Sheep Zebrafish
• Multiple organisms provide suitable systems for experimentation– Zebrafish-development– Rat-physiology– Dog- genetic disease– Fruit fly- behavior
• Some of practical significance– Mosquito/Malaria parasite
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Il metodo Sanger (o metodo a terminazione di catena)
Nel sequenziamento a terminazione di catena la reazione oltre ad
1) Uno stampo a singola elica
2) un innesco specifico (primer)
3) La marcatura con un dNTP* marcato, di solito con 35S
ha bisogno di:
4) una miscela di ddNTP, uno per ogni reazione di sequenza
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La sintesi del filamento compementare, tuttavia non prosegueindefinitivamente, perché la miscela di reazione contiene, in quattro
distinte reazioni piccole quantità di specifici dideossinucleotiditrifosfati, ddATP, ddTTP, ddCTP e ddGTP, che bloccano l’allungamento perché
posseggono un solo atomo di idrogeno al posto del gruppo -OH in 3’
Terminazione della catena
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DNA stampo a singola elica
3’-GGCTAAC
3’-GGCTAAC5’ 3’
Ibridazione conIl primer
+ [35S]dATP+dCTP,dGTP,dTT (dNTP) +Sequenasi
ddATP, dNTP ddCTP, dNTP ddGTP, dNTP ddTTP, dNTP
-CCG ddA -ddC-C ddC
-CC ddG-CCGATT ddG
-CCGA ddT-CCGAT ddT
Sequenza: 5’-CCGATTG
A C G T
-CCGATT ddG-CCGAT ddT-CCGA ddT-CCG ddA-CC ddG-C ddC-ddC
GT
T
AGCC
Schema di sequenziamento a terminazione di catena
Direzione dilettura
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Il sequenziamento automatizzato con marcatori fluorescenti
Coniugando a ciascun ddNTPun diverso marcatore fluorescente, è
possibile effettuare le quattro reazioni di sequenziamento in un unico tubo da saggio
e caricare il tutto in solo pozzetto di gel
ddA
ddC
ddT
ddG
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Le emissioni fluorescenti vengono captate da un rilevatore e leinformazioni vengono integrate e trasformate in picchi di colore diverso, con aree proporzionali all’intensità di emissione.
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Fluorescent DNA Sequencing
A G T A C T G G G A T C
GelElectrophoresis
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Detection of Fluorescently Tagged DNA
DNA FragmentsSeparated by
Electrophoresis
Output to Computer
Scanning Laser Excites
Fluorescent Dyes
Optical Detection System
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Fluorescent DNA Sequencing Data
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Fluorescent DNA Sequence Trace
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Size of gene library
N = ln(1-P) ln (1-A/B)
N = Number of clones P = 95 % probability of finding geneA = Average size of DNA fragmentsB = Total size of genome
E. coli has genome of 4,800,000 nucleotidesAverage size of insert is 10,000 nucleotidesNumber of clones for 95 % probability is 1700
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Size of gene library
• If genome is large e.g. human genome (3 x 109) then number of clones to make library becomes unrealistic (1058000) if using a plasmid vector (accepts only 10 kb as larger DNA can’t be transformed)
• Therefore need to use vectors that can accept larger pieces of DNA– I.e. if each vector contains a large piece of DNA
you don’t need so many clones to make a gene library
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What vectors for what libraries?
VectorInsertsizeWhat librariesPlasmid<10 kbBacteriaLambda phage18-25 kbYeastCosmid34-45 kbIntermediateeukaryotesYAC etc0.1 – 1 MbHigher Eukaryotes
Human library requires 14000 YAC clones
Human library requires >1,000,000 plasmid clones
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Vettori e clonaggio
YAC=yeast artificial chromosome
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Vettori e clonaggio
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Vettori e clonaggio
Lunghezza del DNA
clonabile in uno YAC:
fino a 1000 Kb
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Vettori e clonaggio
Approximate Maximum Length of DNA That Can Be Cloned in Vectors
Vector Type Cloned DNA (kb)
Plasmid
λ phage
Cosmid
P1 phage
BAC (bacterial artificial chromosome)
YAC (yeast artificial chromosome)
20
25
45
100
300
1000
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Whole Genome ShotgunWhole Genome Shotgun • Celera Genomics
Fragment and sequence entire genome
Each fragment is sequenced completely and then these pieces are aligned to one another by a computer, based on overlapping sequence between fragments.
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Overview of Facts Asserted
• 2.91 billion base pairs (bp)
• 1.1% exons, 24% introns, 75% intergenic DNA
• 2.1 million single nucleotide polymorphisms (SNP’s)
• less than 1% of all SNP’s reflected changes in proteins
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Structure of the genome
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http://genome.ucsc.edu
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http://www.ncbi.nlm.nih.gov
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