Biotech Tools Review 2005-2006 1

Biotech Tools Review Recombinant DNA / Cloning gene DNA library restriction enzyme, plasmids, bacteria DNA library cDNA library DNA hybridization Gel electrophoresis RFLP Southern blotting PCR Sanger sequencing Microarray 2005-2006

You want to produce human insulin cheaply for diabetics Find your gene collect mRNA from pancreas cells What to do with the mRNA? 2005-2006 3

You want to produce human insulin cheaply for diabetics What to do with the mRNA? make DNA mRNA  cDNA reverse transcriptase insert cDNA into bacteria using plasmids restriction enzyme ligase transformation How do you find which bacteria carry the recombinant plasmids? 2005-2006 4

You want to produce human insulin cheaply for diabetics How do you find which bacteria carry the recombinant plasmid? amp resistance gene on plasmid LacZ gene plasmid plasmid amp resistance LacZ gene EcoRI BamHI HindIII Which colonies do we want? White ones!! 2005-2006 5

You want to produce human insulin cheaply for diabetics How do you find which bacteria carry the insulin gene on the plasmid? make an insulin gene probe tagged probe hybridization 5’ 3’ labeled probe genomic DNA G A T C 2005-2006 6

What if you want to find out the full sequence of the human insulin gene? Sanger sequencing dideoxynucleotides 2005-2006 7

What if you want to make a lot of copies of your sequenced gene? PCR thermocycler primers Taq polymerase 2005-2006 8

You want to see if 2 people have differences in their insulin genes? Gel electrophoresis & RFLP restriction digest run samples on gel see if there is a polymorphism 2005-2006 9

What if I want to locate a sequence on an electrophoresis gel? Southern blot tagged probe 2005-2006 10

You want to test effect of drug on gene expression in pancreas? Microarray collect mRNA mRNA  cDNA hybridize on GeneChip 2005-2006 11

Any Questions?? 2005-2006 12