Recombinant DNA Technology

BY- Dr. Armaan Singh

Cloning Vector

Required features

1. Origin of replication

2. Selectable marker

3. Screenable marker for recombinant molecules

4. Cloning sites

Restriction Modification System

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA* *

AAGATGCGAATTCGTACA* *

DNA methylase

Restriction endonuclease

AAGATGCGAATTCGTACA

AAGATGCGAATTCGTACA

AAGATGCG AATTCGTACA

DNA methylase

Restriction endonuclease

5’ ATGCGAATTCCGGTT 3’3’ TACGCTTAAGGCCTT 5’

5’-ATGCG-3’ 5’-AATTCCGGTT-3’3’-TACGCTTAA-5’ 3’-GGCCTT-5’

EcoR1

5’ ATGCGATATCCGGTT 3’3’ TACGCTATAGGCCTT 5’

5’-ATGCGAT-3’ 5’-ATCCGGTT-3’3’-TACGCTA-5’ 3’-TAGGCCTT-5’

EcoRV

Sticky-end cutter

Blunt-end cutter

T4 DNA Ligase

Transformation• All of the previous steps were

performed in vitro. • We have generated a very small

amount of a recombinant plasmid• Need to amplify in bacteria to get

enough to work with. • Transformation – process to mobilize

DNA into bacterial host• Select for transformed bacteria on

specific antibiotic that corresponds to the antibiotic resistance gene present on the plasmid

How to produce a recombinant protein

10 to 70% of cellular protein

0.1 to 1% of cellular protein

cDNA

cDNA Library

cDNA

DNA hydridization screening for specific gene

•Requires that you know something about the gene sequence•Can get sequence information form purified protein

Product name  Protein type Application Company

Adagen (Adenosine deaminase )

An enzyme Severe combined immunodeficiency disease (SCID)

Enzon

Genotropin (Recombinant

growth hormone)

A hormone Growth hormone deficiency (GHD) in

children

Pharmacia & Upjohn

Humalog (Recombinant human

insulin)

A hormone Diabetes Eli Lilly

Nabi-HB (Anti-Hepatitis B)

 An antibody Hepatitis-B  Nabi

Novo Seven (Recombinant

coagulation factor VIIa)

A modified factor Hemophillia patients with inhibitors

Novo Nordisk

Ontak (Diphtheria toxin-interleukin-2)

A fusion protein Cutaneous T-cell lymphoma (CTCL)

Ligand Pharmaceuticals

Roferon-A (Recombinant

interferon alfa-2a)

A modifier Hairy cell leukemia or AIDS-related Kaposi's sarcoma

Hoffmann-La Roche

Genetic Modification of Higher Organisms• Can introduce gene into animals and plants• These modified organism are powerful research

tools to study the effect of a specific gene product on metabolism, development etc….• Has also been used to develop improved

agricultural products

Genetically Engineered SalmonIs Bigger Better?

http://www.agwest.sk.ca/sabic_index_tp.shtml