cloning:recombinant dna multistep process . produce fragments of dna using enzymes that cut dna at...
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Cloning:Recombinant DNACloning:Recombinant DNA
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Multistep ProcessMultistep Process
. Produce fragments of DNA using enzymes that cut DNA at specific base sequences.
. Link these fragments to self-replicating forms of DNA = vectors.
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. Replicate the recombinant DNA molecule in the host organism (1000’s of copies).
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. Retrieve the cloned copies for use or modification.
. Produce and purify gene product.
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Some useful definitionsSome useful definitions
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Restriction EnzymesRestriction Enzymes
Enzymes that recognize a specific base sequence in DNA and cleave at that site
Isolated from bacteria that inactivated viruses via cutting their DNA
“Molecular scissors”
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Recognition sequenceRecognition sequence
Palindrome - sequence is read the same on either strand, when read from 5’ to 3’
Creates either sticky ends or blunt ends
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Eco R1Eco R1
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VectorsVectors
A self-replicating DNA molecule that is used to transfer foreign DNA fragments between cells.
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Steps in CloningSteps in Cloning
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Steps in cloning - GeneralSteps in cloning - General
Isolate vector DNA and gene of interest
Cut both with the same restriction enzyme
Mix DNA’s and ligate = recombinant DNA
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Transfer recombinant molecule into host cell (transform)
Grow/Select transformants
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Types of Vectors and DNA Types of Vectors and DNA delivery systemsdelivery systems
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Types of VectorsTypes of Vectors
PlasmidPhage (virus)CosmidYeast Artificial Chromosome
(YAC)
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PlasmidsPlasmids
Circular extrachromosomal DNA molecules naturally found in bacteria
Self-replicatingCan insert pieces up to 10kb
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Plasmid vectors need…Plasmid vectors need…
origin of replicationselectable marker (antibiotic)unique restriction enzyme
cleavage sites
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Plasmid Placement in CellPlasmid Placement in Cell
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Phage vectorsPhage vectors
Derivatives of phage (lambda)
Linear DNACan insert up to 15 kb
fragments
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Phage InsertionPhage Insertion
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CosmidsCosmids
Don’t occur naturallyConstructed using features of
both plasmids and phage Can carry inserts up to 45 kb
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YACsYACs
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YACsYACs
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YACsYACs
Yeast artificial chromosomeSelf-replicating elementsCan insert segments up to 1
million base pairsCan replicate any inserted DNA
via transfer to yeast cells
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Essential elements for YACsEssential elements for YACs
Tel - telomeresCen - centromereOri - Origin of replicationSelectable markersRestriction enzyme recognition
sites
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Particle GunParticle Gun
Usually using cell cultureShoot DNA coated objects into
cellsTungsten pellets, Whiskers
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We can insert the gene into We can insert the gene into cells – Now what?cells – Now what?
Selecting for transformed cells and amplifying the
product
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Basic StepsBasic Steps
Identify the transformantsIsolate transformed coloniesAmplify the product
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Identifying transformantsIdentifying transformants
Vectors containing antibiotic resistance genes can be used
Those that took up the vector will now express antibiotic resistance
Ability to metabolize substances included in media
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Isolate Colonies of InterestIsolate Colonies of Interest
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Amplify the ProductAmplify the Product
Use bacteria (usually E. Coli) to amplify product
Sometimes yeast cells, if the gene you are amplifying is a eukaryote specific gene
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Genetic LibrariesGenetic Libraries
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Genetic libraryGenetic library
Collection of clones that contains all the genetic information of an individual = genomic library - gene bank
Chromosomes, set of genes of single cell type etc.
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cDNA - mRNA population made into cDNA. Produce clones
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Can recover genes of interest from libraries for–Clinical studies–Evolutionary comparison–Experimental studies–Commercial use
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Construction of...Construction of...
DNA isolated from an organismDigest into smaller segments
which can be inserted within vectors (size limitations)
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record of genome or portion ofCan be screened, hybridization
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