Genetic Engineering Dr Swati Paliwal Assistant Professor Department

Genetic Engineering Dr. Swati Paliwal Assistant Professor Department of Bio Science & Bio Technology Banasthali Vidyapith Rajasthan

Genetic Engineering • Recombinant DNA technology – Mixing DNA – No species limitations • Multiple species – Novel genes • • Produce natural proteins Invent new proteins Manipulate organisms Disease treatment

Protein production • Diabetes type I – Defective insulin genes – Autoimmune attack – insulin • Pituitary dwarfism type I – Defective growth hormone (GH 1) – GH 1 from human cadavers→ Creutzfeld-Jacob disease – Safe supply needed

Expression Vector • Plasmid Antibiotic resistance

• Restriction enzymes Bacterial enzymes – Foreign DNA restricted • Chop DNA at specific sequences – Restriction sites – Palidromes • Level, 1234321 …GAATTC… …CTTAAG… – Cuts can leave: sticky ends or …G AATTC… …CTTAA G… blunt ends …GAA …CTT TTC… AAG…

Fig 19. 2

Transformation Growth Medium + Antibiotic resistance

• Chemical Transformation – Ca++ – Specialized lipids • Lipofectamine • Electroporation – ~1000 Volts for 5 -10 ms – Creates pores

Protein production Sample Positive clones Antibiotic resistance Grow Extract protein (GH or insulin) m. RNA → c. DNA No introns Confirm protein identity Human gene produced by bacteria Treat

Experimental techniques • Gel electrophoresis – Molecular separation – Gel • Agarose – polysaccharide • Polyacrylamide • Like a: – Sponge – Sieve

Sanger sequencing

Gene therapy • Ex) SCID – Severe combined immune deficiency – T cell disease • Problem – Random gene insertion Fig 19. 15

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