Transcript DNA analysis - Madeira City Schools

We’re going to look at the following:
 DNA analysis: this makes it possible to examine
variation among individuals. Why would you want to
do this?
 Genetic engineering: manipulating DNA or
organisms to perform practical tasks or provide
useful products
I. DNA analysis:
Result of an electrophoresis
A. Most common uses are:
1. analyze a person’s genes (looking for diseases)
2. compare the sequence of nitrogen bases among
individuals (paternity and crime scenes)
B. Use restriction enzymes
1. A restriction enzyme is an enzyme that cuts DNA at specific
“recognition sites”
2. In nature restriction enzymes protect bacteria against
intruding DNA (viruses) by cutting the intruders DNA (can’t
function)
C. Use an electrophoresis apparatus to separate the cut DNA
1. Fancy way to do chromatography
D. Process of DNA analysis
1. DNA is obtained (hair, blood, skin cells…)
2. Copies of DNA are made using the PCR method
(polymerase chain reaction). This is just a complex way to make a ton of DNA copies
3. Restriction enzymes are added to cut the DNA into
fragments at very specific sequences.
4. DNA has an overall negative charge
5. Put DNA into wells of gel at the negative end of the
chamber
6. Turn on power supply
7. Smaller fragments will travel faster
through gel, therefore they will
travel the farthest in the allotted time.
Gel that is loaded with DNA and reading to “run”
No
DNA in
these
wells
DNA is
present
in these
wells
II. Genetic Engineering
A. An overview:
1. Use bacteria plasmids - small circular DNA that
replicate within the bacterial cell. These are isolated.
2. The plasmid and gene of choice are both cut using the same restriction
enzyme (therefore cutting at the same recognition site)
b. this produces what we call “sticky ends”
3. The plasmid and gene of choice are put in a test tube together
4. DNA ligase (an enzyme) is added fuse the two strands together
5. Plasmid is put back into bacteria
6. Bacteria reproduces
7. Bacteria will express the inserted gene by making the protein
GAATTC
CTTAAG
G
CTTAA
AATTC
G
B. What you will be doing in your lab:
1. Gene from a bioluminescent jellyfish has been isolated
a. it produces a green fluorescent protein (GFP)
2. It has been inserted into a bacterial plasmid.
a. the recombinant DNA molecule is called “pGLO” plasmid
3. The plasmid will (hopefully) transform into bacteria that will
then express the inserted gene and therefore glow.
a. Genetic transformation = change caused by genes
C. Things you need to know to understand what you are doing
1. Inserting the gene is not enough…you have to have
something to turn it on
2. Turning on and off genes (gene expression) is carefully
regulated to allow for adaptation to different conditions
a. prevents wasteful production of unneeded proteins
b. digestive enzymes are highly regulated
3. Bacteria regulates their gene expression through “operons”
a. an operon is a cluster of genes controlled by one
promoter
The Arabinose Operon
araC
araB
araA
araD
OFF
araC
araB
araA
araD
ON
The pGLO plasmid
araC
GFP
ON
Glowing
4. An ampicillin resistant gene is also added to your pGLO
plasmid
a. this is a gene that makes a protein that allows the
bacteria to grow in the presence of the antibiotic
therefore making it “resistant”
ampicillin,
b. This insures that only the bacteria that was transformed
will grow…nothing else.
c. It is a way to control your experiment and not add more
variables.
GFP – makes
Origin – where
transcription starts
The pGLO
plasmid
green
flourescent
protein
Bla gene – makes bacteria resistant to
ampicillin
What you will see in lab:
LB = Luria broth (food for bacteria)
AMP = Ampicillin (antibiotic -- kills bacteria)
ARA= Arabinose (sugar -- turns on the pGLO gene)
LB
LB/AMP
LB/AMP
LB/AMP/
ARA
Plasmid added
---
---
+
+
Purpose
Control for
bacterial
growth
Control for
effectiveness
of ampicillin
Control for
effectiveness
of
transformation
End result
Does bacteria
gRow
Does bacteria
gLow