• Enzymatic digesting
– Endonuclease
digestion: site specific
– Restriction
Sequence specific binding Endonucleases
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Restriction Endonuclease Recombinant DNA Technology
Digestion Cutting and Pasting DNA
Enzymology
Cutting
Restriction Pasting
Endonuclease DNA Ligase
Digests DNA ligates DNA
Proteins (Enzymes) can cut and paste DNA
Three Steps
Restriction • 1. Cut source and vector DNA
Endonuclease
cuts DNA – Restriction Endonuclease Digestion
• Hybridization (nonenzymatic)
Vector DNA – Sticky ends (complementary and antiparallel)
– Origin of replication – Salt and temperature
– Capable of independent replication in a
host • Ligation (enzymatic)
– Capable of incorporating DNA – DNA ligase – create phosphodiester bonds complete
– DNA sequence contains unique restriction covalently joined sugar-phosphate backbones
site
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Engineering Recombinant DNA Recombinant DNA Technology
Copying DNA
Vector DNA
Source DNA
Plasmids
• Small circular
• Many copies per cell
• Replicate independently • Means of identifying the recombinant vector
• Convenient restriction sites • Means of recovery of recombinant vector
• Unique (single cut) restriction sites • Choice depends on size of insert
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Vectors
Bacteriophage vectors
• Single stranded
• Double stranded
• Size of insert limited
• Dispensible sequence can
be replaced with insert
sequence
• Headful packaging limits
insert size