Recombinant DNARecombinant DNA

ScopeScopeHuman Genome = 3x10Human Genome = 3x1099

Average Gene = 3x10Average Gene = 3x104 4 (1/10(1/1055))

SNP Mutation (1/10SNP Mutation (1/1099))

ProcessProcessCut DNA into piecesCut DNA into piecesInsert DNA into vectors that can replicate in Insert DNA into vectors that can replicate in bacteriabacteriaTransform (introduce) DNA into host cellTransform (introduce) DNA into host cellPlate cells and select those with vectorsPlate cells and select those with vectorsEach colony has one chunk of DNAEach colony has one chunk of DNAThe whole set is a library of human DNAThe whole set is a library of human DNA

SpecificsSpecificsRestriction enzyme Restriction enzyme GAATTCGAATTC

PalindromePalindromeExists for a large Exists for a large number of locinumber of loci

Ligase can bind together Ligase can bind together DNA from any organism DNA from any organism that has been cut with that has been cut with the enzymethe enzyme

Comes from bacteria Comes from bacteria that need to repair their that need to repair their own DNAown DNA

Hook fragment to Hook fragment to bacterial DNA with a bacterial DNA with a replication site.replication site.

Selecting CellsSelecting Cells

EpisomesEpisomesContain resistance genesContain resistance genes

Are transferred between bacteria to Are transferred between bacteria to allow for more rapid adaptationallow for more rapid adaptation

We can insert all of the fragments from We can insert all of the fragments from the human genome into bacterial the human genome into bacterial episomes and create a library of episomes and create a library of bacterial colonies that can be used to bacterial colonies that can be used to replicate DNAreplicate DNA

Reverse Reverse TranslationTranslation

You can take a protein like beta-globin You can take a protein like beta-globin and determine the amino acid sequenceand determine the amino acid sequence

You can then create a set of You can then create a set of complimentary probes for all possible complimentary probes for all possible nucleotide sequences that would cause nucleotide sequences that would cause the amino acid sequencethe amino acid sequence

Dump the probe on each entry in the Dump the probe on each entry in the library to find the gene that makes library to find the gene that makes beta-globinbeta-globin

Other ApproachesOther ApproachesInsert DNA into a virus and infect Insert DNA into a virus and infect malalian cells.malalian cells.

Create artificial chromosomes and inject Create artificial chromosomes and inject in the nucleusin the nucleus

Bacterial Artificial ChromosomeBacterial Artificial Chromosome

Stem Cell ManipulationStem Cell Manipulation

Lets grow insulinLets grow insulinInsert insulin gene into Insert insulin gene into plasmidplasmid

But promoters for e-coli are But promoters for e-coli are different than humandifferent than human

So put e-coli promoter on So put e-coli promoter on the front of episomethe front of episome

But humans do splicing and But humans do splicing and bacteria don’tbacteria don’t

So, we need to insert So, we need to insert spliced gene into epiisomespliced gene into epiisomeUse reverse transcriptase Use reverse transcriptase to turn RNA into DNAto turn RNA into DNA

Viruses use this to Viruses use this to replicatereplicate

But protein may fold But protein may fold differently in bacteria, so differently in bacteria, so you may have to do some you may have to do some more engineeringmore engineering

How about How about mammalsmammals

Rabies antibodies are importantRabies antibodies are important

It is hard to find people that generate themIt is hard to find people that generate them

People don’t like to be infected to generate People don’t like to be infected to generate antibodies for pharma companiesantibodies for pharma companies

Put human gene that generates antibody into Put human gene that generates antibody into mouse, infect it and collect antibodiesmouse, infect it and collect antibodies

But mice don’t have much blood, lets do it with a cowBut mice don’t have much blood, lets do it with a cow

But the cows immune system also generates cow But the cows immune system also generates cow antibodiesantibodies

So lets cut out the cow immune system genes and So lets cut out the cow immune system genes and totally replace them with human immune systemtotally replace them with human immune systemHope that the cow doesn’t get sickHope that the cow doesn’t get sick