Recombinant DNA

1
Recombinant DNA
2
Scope

• Human Genome 3x109
• Average Gene 3x104 (1/105)
• SNP Mutation (1/109)
• 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
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

5
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

6
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

7
Lets grow insulin

• Insert insulin gene into episome
• 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 dont
• 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

8
How about mammals

• Rabies antibodies are important
• It is hard to find people that generate them
• People dont 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 dont 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 doesnt get sick

9
(No Transcript)