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2005-2006 AP Biology
Biotech Tools Review
2005-2006AP 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
2005-2006AP Biology
You want to produce human insulin cheaply for diabetics Find your gene
collect mRNA from pancreas cells
What to do with the mRNA?
2005-2006AP 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?
2005-2006AP 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
ampresistance
LacZ gene
EcoRI
BamHIHindIII
2005-2006AP 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 G A T C A G T A G
C T A G T C A T C
2005-2006AP Biology
What if you want to find out the full sequence of the human insulin gene? Sanger sequencing
dideoxynucleotides
2005-2006AP Biology
What if you want to make a lot of copies of your sequenced gene? PCR
thermocycler primers Taq polymerase
2005-2006AP 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
2005-2006AP Biology
What if I want to locate a sequence on an electrophoresis gel? Southern blot
tagged probe
2005-2006AP Biology
You want to test effect of drug on gene expression in pancreas? Microarray
collect mRNA mRNA cDNA
hybridize on GeneChip
2005-2006 AP Biology
Any Questions??