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