Cutting DNACutting DNA

Restriction endonucleases Restriction endonucleases (restriction enzymes)(restriction enzymes)• sticky endssticky ends• blunt endsblunt ends

NomenclatureNomenclature• EcoEcoRIRI• EE = genus ( = genus (EscherichiaEscherichia))• coco = species ( = species (colicoli))• R = strainR = strain• I = # of enzymeI = # of enzyme

Blunt & Sticky ends

Pasting DNAPasting DNA

Complementary ends Complementary ends (sticky ends) H-bond(sticky ends) H-bond

Ligase forms Ligase forms phosphodiester bond phosphodiester bond to seal strands to seal strands together.together.

1. Contains an origin of replication, allowing for replication independent of host’s genome.

2. Contains Selective markers: Selection of cells containing a plasmid twin antibiotic resistanceblue-white screening

3. Contains a multiple cloning site (MCS)4. Easy to be isolated from the host cell.

A plasmid vector for cloning A plasmid vector for cloning

Plasmid Plasmid vectorsvectors

Expression Expression vectorvector

How to clone DNAHow to clone DNA Isolation of cloning vector Isolation of cloning vector

(bacterial plasmid) & gene-(bacterial plasmid) & gene-source DNA (gene of interest)source DNA (gene of interest)

Insertion of gene-source DNA Insertion of gene-source DNA into the cloning vector using into the cloning vector using the same restriction enzyme; the same restriction enzyme; bind the fragmented DNA with bind the fragmented DNA with DNA ligaseDNA ligase

Introduction of cloning vector Introduction of cloning vector into cells (transformation by into cells (transformation by bacterial cells)bacterial cells)

Cloning of cells (and foreign Cloning of cells (and foreign genes)genes)

Identification of cell clones Identification of cell clones carrying the gene of interestcarrying the gene of interest

Screening of the cloneScreening of the clone

The medium in this petri The medium in this petri dish contains the dish contains the antibiotic Kanamycinantibiotic Kanamycin

The bacteria on the right The bacteria on the right contain Kanr, a plasmid contain Kanr, a plasmid that is resistant to that is resistant to Kanamycin, while the Kanamycin, while the one on the left has no one on the left has no resistanceresistance

Note the difference in Note the difference in growthgrowth

Blue/White Color Blue/White Color ScreeningScreening

lacZ lacZ insert

functional enzyme nonfunctional enzyme

X-gal product X-gal product

Selecting Colonies with Selecting Colonies with Recombinant PlasmidsRecombinant Plasmids