Transcript Advanced Techniques

Biotech Tools Review
AP Biology
Modified from: Kim Foglia, Explore 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

EcoRI
BamHI
HindIII
LacZ gene
plasmid
amp
resistance
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
labeled probe
genomic DNA
3’
AP Biology
G A T C AG T A G
CTAGTCAT C
5’
What if you want to find out the full
sequence of the human insulin gene?
 Sanger sequencing

AP Biology
dideoxynucleotides
What if you want to make a lot of
copies of your sequenced gene?
 PCR



AP Biology
thermocycler
primers
Taq polymerase
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

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tagged probe
You want to test effect of drug on
gene expression in pancreas?
 Microarray
collect mRNA
 mRNA  cDNA

 hybridize on GeneChip
AP Biology
Any Questions??
AP Biology
Modified from: Kim Foglia, Explore Biology