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??