Cloning Recombinant DNA Multistep Process Produce fragments of
Cloning: Recombinant DNA
Multistep Process. Produce fragments of DNA using enzymes that cut DNA at specific base sequences. 2. Link these fragments to selfreplicating forms of DNA = vectors. 1
3 . Replicate the recombinant DNA molecule in the host organism (1000’s of copies).
. Retrieve the cloned copies for use or modification. 5. Produce and purify gene product. 4
Some useful definitions
Restriction Enzymes l Enzymes that recognize a specific base sequence in DNA and cleave at that site l Isolated from bacteria that inactivated viruses via cutting their DNA l “Molecular scissors”
Recognition sequence l Palindrome - sequence is read the same on either strand, when read from 5’ to 3’ l Creates either sticky ends or blunt ends
Eco R 1
Vectors l. A self-replicating DNA molecule that is used to transfer foreign DNA fragments between cells.
Steps in Cloning
Steps in cloning - General l Isolate vector DNA and gene of interest l Cut both with the same restriction enzyme l Mix DNA’s and ligate = recombinant DNA
l Transfer recombinant molecule into host cell (transform) l Grow/Select transformants
Types of Vectors and DNA delivery systems
Types of Vectors l Plasmid l Phage (virus) l Cosmid l Yeast Artificial Chromosome (YAC)
Plasmids l Circular extrachromosomal DNA molecules naturally found in bacteria l Self-replicating l Can insert pieces up to 10 kb
Plasmid vectors need… l origin of replication l selectable marker (antibiotic) l unique restriction enzyme cleavage sites
Plasmid Placement in Cell
Phage vectors l Derivatives l Linear of phage (lambda) DNA l Can insert up to 15 kb fragments
Phage Insertion
Cosmids l Don’t occur naturally l Constructed using features of both plasmids and phage l Can carry inserts up to 45 kb
YACs
YACs
YACs l Yeast artificial chromosome l Self-replicating elements l Can insert segments up to 1 million base pairs l Can replicate any inserted DNA via transfer to yeast cells
Essential elements for YACs l Tel - telomeres l Cen - centromere l Ori - Origin of replication l Selectable markers l Restriction enzyme recognition sites
Particle Gun l Usually using cell culture l Shoot DNA coated objects into cells l Tungsten pellets, Whiskers
We can insert the gene into cells – Now what? Selecting for transformed cells and amplifying the product
Basic Steps l Identify the transformants l Isolate transformed colonies l Amplify the product
Identifying transformants l Vectors containing antibiotic resistance genes can be used l Those that took up the vector will now express antibiotic resistance l Ability to metabolize substances included in media
Isolate Colonies of Interest
Amplify the Product l Use bacteria (usually E. Coli) to amplify product l Sometimes yeast cells, if the gene you are amplifying is a eukaryote specific gene
Genetic Libraries
Genetic library l Collection of clones that contains all the genetic information of an individual = genomic library - gene bank l Chromosomes, set of genes of single cell type etc.
l c. DNA - m. RNA population made into c. DNA. Produce clones
l Can recover genes of interest from libraries for – Clinical studies – Evolutionary comparison – Experimental studies – Commercial use
Construction of. . . l DNA isolated from an organism l Digest into smaller segments which can be inserted within vectors (size limitations)
l record of genome or portion of l Can be screened, hybridization
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