1. Recombinant DNA

2. Scope Human Genome = 3x10 9 Average Gene = 3x10 4 (1/10 5 ) SNP Mutation (1/10 9 ) Process Cut DNA into pieces Insert DNA into vectors that can replicate in bacteria Transform (introduce) DNA into host cell Plate cells and select those with vectors Each colony has one chunk of DNA The whole set is a library of human DNA

3. Specifics Restriction enzyme GAATTC Palindrome Exists for a large number of loci Ligase can bind together DNA from any organism that has been cut with the enzyme Comes from bacteria that need to repair their own DNA Hook fragment to bacterial DNA with a replication site.

4. Selecting Cells

5. Episomes Contain resistance genes Are transferred between bacteria to allow for more rapid adaptation We can insert all of the fragments from the human genome into bacterial episomes and create a library of bacterial colonies that can be used to replicate DNA

6. Reverse Translation You can take a protein like beta-globin and determine the amino acid sequence You can then create a set of complimentary probes for all possible nucleotide sequences that would cause the amino acid sequence Dump the probe on each entry in the library to find the gene that makes beta-globin

7. Other Approaches Insert DNA into a virus and infect malalian cells. Create artificial chromosomes and inject in the nucleus Bacterial Artificial Chromosome Stem Cell Manipulation

8. Lets grow insulin Insert insulin gene into plasmid But promoters for e-coli are different than human So put e-coli promoter on the front of episome But humans do splicing and bacteria don’t So, we need to insert spliced gene into epiisome Use reverse transcriptase to turn RNA into DNA Viruses use this to replicate But protein may fold differently in bacteria, so you may have to do some more engineering

9. How about mammals Rabies antibodies are important It is hard to find people that generate them People don’t like to be infected to generate antibodies for pharma companies Put human gene that generates antibody into mouse, infect it and collect antibodies But mice don’t have much blood, lets do it with a cow But the cows immune system also generates cow antibodies So lets cut out the cow immune system genes and totally replace them with human immune system Hope that the cow doesn’t get sick