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Restriction Enzymes
• Restriction enzymes cut
DNA from any source
into fragments
• Sticky ends can pair up
with complementary
DNA with the help of
ligase producing
recombinant DNA
• Example: EcoRI, HindII
2. Must Be Able To Insert Gene
Of Interest (GOI) Into A
Vector
• Vector: plasmid or
virus
– Plasmid: small circular
double stranded DNA
– Carries only a few genes
– Marker:antibiotic
resistance
PCR—Polymerase Chain
Reaction
•
•
•
Denaturation at 94º C  DNA
disassociates to SS
Add annealing primers at 54 º C :enables
polymerase to attach
Extensions at ~72 º C :taq polymerase &
lots of nucleotides
PCR
• Steps 1-3 are repeated
many times
• A single fragment of
DNA produces
– > 1 million copies!
– A few hours = 100 billion
copies!
• Tiny speck of blood and
amplify it for crime lab