Cloning:Recombinant DNA Multistep Process . Produce fragments of DNA using enzymes that cut DNA at...

Cloning:Recombinant DNACloning:Recombinant DNA

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.

. Replicate the recombinant DNA molecule in the host organism (1000’s of copies).

. Retrieve the cloned copies for use or modification.

. Produce and purify gene product.

Some useful definitionsSome useful definitions

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”

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

Eco R1Eco R1

VectorsVectors

A self-replicating DNA molecule that is used to transfer foreign DNA fragments between cells.

Steps in CloningSteps in Cloning

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

Transfer recombinant molecule into host cell (transform)

Grow/Select transformants

Types of Vectors and DNA Types of Vectors and DNA delivery systemsdelivery systems

Types of VectorsTypes of Vectors

PlasmidPhage (virus)CosmidYeast Artificial Chromosome

(YAC)

PlasmidsPlasmids

Circular extrachromosomal DNA molecules naturally found in bacteria

Self-replicatingCan insert pieces up to 10kb

Plasmid vectors need…Plasmid vectors need…

origin of replicationselectable marker (antibiotic)unique restriction enzyme

cleavage sites

Plasmid Placement in CellPlasmid Placement in Cell

Phage vectorsPhage vectors

Derivatives of phage (lambda)

Linear DNACan insert up to 15 kb

fragments

Phage InsertionPhage Insertion

CosmidsCosmids

Don’t occur naturallyConstructed using features of

both plasmids and phage Can carry inserts up to 45 kb

YACsYACs

YACsYACs

YACsYACs

Yeast artificial chromosomeSelf-replicating elementsCan insert segments up to 1

million base pairsCan replicate any inserted DNA

via transfer to yeast cells

Essential elements for YACsEssential elements for YACs

Tel - telomeresCen - centromereOri - Origin of replicationSelectable markersRestriction enzyme recognition

sites

Particle GunParticle Gun

Usually using cell cultureShoot DNA coated objects into

cellsTungsten pellets, Whiskers

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

Basic StepsBasic Steps

Identify the transformantsIsolate transformed coloniesAmplify the product

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

Isolate Colonies of InterestIsolate Colonies of Interest

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

Genetic LibrariesGenetic Libraries

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.

cDNA - mRNA population made into cDNA. Produce clones

Can recover genes of interest from libraries for–Clinical studies–Evolutionary comparison–Experimental studies–Commercial use

Construction of...Construction of...

DNA isolated from an organismDigest into smaller segments

which can be inserted within vectors (size limitations)

record of genome or portion ofCan be screened, hybridization