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Introduction Tools Process Applications

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Introduction – basic idea– Gene is identified and excised from

one organism– Gene is placed in vector (plasmid)

and amplified– Gene is transferred to new organism

or used in host organism to make protein product

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Restriction endonucleases– Nucleases cut nucleic acid – at first

seem non specific– Linn & Abner discover that some

strains of bacteria are able to resist bacteriophage infection by digesting infecting DNA

– Different bacteria produce different restriction enzymes

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Restriction Endonucleases– Cut at specific 4, 6, or 8 base sites– Site is a “palindrome”

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– Some restriction enzymes generate “sticky ends”

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Plasmids– Carry an antibiotic resistance marker– Carry restriction sites in convenient

locations to insert DNA– Carry characteristics that allow the

plasmid to reproduce in several organisms

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Polymerase Chain Reaction (PCR)– Allows any segment of DNA to be

amplified chemically in minutes– Uses a thermostable DNA polymerase– Machine can cycle every 60-90

seconds

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Agarose Gel Electrophoresis– Can separate DNA fragments made

with restriction enzymes– Can separate PCR made DNA– Can be used to sequence DNA

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Basic process worked out by Cohen & Boyer

Cut plasmid DNA and target DNA with same restriction enzyme

Mix DNA and allow sticky ends to match up

Select DNA clones having target gene

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