AP Biology Biotech Tools Review

AP Biology Biotech Tools Review  Recombinant DNA / Cloning gene  restriction enzyme, plasmids, bacteria  DNA library  cDNA library  DNA hybridization  Gel electrophoresis  RFLP  Southern blotting  PCR  Sanger sequencing  Microarray

AP Biology You want to produce human insulin cheaply for diabetics  Find your gene  collect mRNA from pancreas cells  What to do with the mRNA?

AP Biology 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?

AP Biology 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

AP Biology 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 GATCAGTAG CTAGTCATC

AP Biology What if you want to find out the full sequence of the human insulin gene?  Sanger sequencing  dideoxynucleotides

AP Biology What if you want to make a lot of copies of your sequenced gene?  PCR  thermocycler  primers  Taq polymerase

AP Biology 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

AP Biology What if I want to locate a sequence on an electrophoresis gel?  Southern blot  tagged probe

AP Biology You want to test effect of drug on gene expression in pancreas?  Microarray  collect mRNA  mRNA  cDNA  hybridize on GeneChip

AP Biology Any Questions??