Recombinant DNA technology Pierce GENETICA Zanichelli editore S

• The objectives of Recombinant DNA technology include: – Identifying genes – Isolating genes – Modifying genes – Re-expressing genes in other hosts or organisms Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

• These steps permit scientists and clinicians to: – Identify new genes and the proteins they encode – Mutate genes – Correct endogenous genetic defects – Manufacture large quantities of specific genes (hormones, vaccines, and other biological agents of medical interest) Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

The strategy Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Genomic libraries Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

c. DNA libraries Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Restriction cloning Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Tailing Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Linkers addition Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Different cuts of restriction enzymes • Blunt ends Es. Sma. I • 5' protruding Es. Eco. RI • 3' protruding Es. Pst. I 5'-CCCGGG-3' 3'-GGGCCC-5' 5'-GAATTC-3' 3'-CTTAAG-5' 5'-CTGCAG-3' 3'-GACGTC-5' 5'-CCC-3' 3'-GGG-5' 5'-GGG-3' 3'-CCC-5' 5'-G-3' 3'-CTTAA-5' 5'-AATTC-3' 3'-G-5' 5'-CTGCA-3' 3'-G-5' 5'-G-3' 3'-ACGTC-5'

DNA extraction from gel • Ion exchange columns • Electroelution • Gel-filtration • Low

Filling in Nel corso dei clonaggi può capitare di dover trasformare estremità coesive sporgenti al 5' o al 3‘ in estremità piatte, sintetizzando le basi mancanti nel filamento incompleto al 5' (filling in), oppure eliminando quelle sporgenti al 3' (trimming) La tecnica d’elezione consiste nel cosiddetto “ filling in” che consiste nel “riempire” una estremità sporgente al 5' con la Klenow 5'P 3'OH G 3'OH C AATTC d. TTP d. ATP d. GTP 5'P 3'OH G TTAAG C AATTC 3'OH 5'P Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

T/A cloning di prodotti di PCR Most DNA Polymerases have a weak activity adding an A to the 3’ end of the amplified products 5'-P HO-3 A A-3'OH 5'-P Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Vectors PLASMIDS 0, 1 to 10 Kb PHAGES 8 to 22 kb COSMIDS 32

CLONING VECTORS • Origin of replication • Selectable marker • Restriction Enzymes Pierce,

-Inserto genomico fino 500 Kb (in Lievito S. cerevisiae) Pierce, GENETICA, Zanichelli editore S.

Phage vectors

Multiple cloning sites (Polylinker) Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Gene transfer methods Bacterias Electroporation Ca. Cl 2 or Rb. Cl 2 transformation Plants Electroporation Agrobacterium Cannon (gold particles) Protoplast fusion Yeast PEG Electroporation DEAE-destran Animal cells PEI Electroporation DEAE-destran Ca. PO 4 Pierce, GENETICA, Zanichelli editore S. p. A. Copyright © 2005

Protein expression vector