1. Steps to Recombinant DNA 1) Isolate the foreign DNA fragment 2) Attach DNA fragment to a “vehicle” called a Vector 3) Transfer the vector into a host

2. 1. Isolate DNA Cut (cleave) small pieces of DNA using a Restriction Enzyme Restriction enzymes are bacterial proteins that cut DNA in a SPECIFIC nucleotide sequence, called a Recognition Site There are 100’s of Restriction Enzyme

3. Example of Step 1 EX: The section of Firefly DNA that codes for the light producing enzyme is cleaved (cut) using a Restriction Enzyme called EcoRI

4. Restriction enzyme Cut the Firefly DNA Sequence at AATT

5. Sticky Ends Where Restriction Enzymes cut the DNA is called Sticky Ends Sticky Ends WANT to join with DNA again, because part of it has become single stranded

6. Sticky ends

7. 2. Vectors The DNA fragments that have been cut, need to be inserted into a Vector (vehicle) Vector- a way that DNA from another species can be carried into the host cell Vectors can be biological or mechanical

8. Vector Examples Biological Vectors: Viruses and Plasmids Plasmids are small rings of DNA found in a bacterial cell Mechanical Vectors: Micropipette or tiny metal bullet

9. Micropipete

10. 2. Example of Vectors The firefly’s light producing DNA is inserted into a Plasmid

11. Step 3: Transfer into a host The recombined DNA is transferred into a bacterial cell (Bacteria = HOST) The bacterial cell replicates up to 500 times per cell making copies of the recombinant DNA

12. Each copy that the bacterial cell makes of the recombinant DNA is called a Gene Clone Rejoining the DNA Fragments (Firefly’s glow code + the Plasmid’s DNA) is called Gene Splicing