BIO 330 Cell Biology
Laboratory
Spring 2011
Human Angiotensinogen Genotyping Lab
Angiotensinogen is the large precursor molecule which, when cleaved by the enzyme called renin, forms
angiotensin I. Angiotensin I is later cleaved by ACE (angiotensin converting enzyme) to form angiotensin
II (Ang II). AngII is one of the primary regulators of blood pressure, but also has other actions, including
vasculogenesis (formation of new blood vessels). Promoter regions in genes are often involved in the
regulation of gene transcription. In the case of angiotensinogen, a common genetic variant (single
nucleotide polymorphism, SNP) in the promoter region leads to increased expression of
angiotensinogen mRNA, in turn leading to increased AngII protein, resulting in higher blood pressure or
other effects. This promoter region SNP is highly linked to a second SNP in the coding region. Humans
with the Thr235 variant have ~20% higher plasma levels of angiotensinogen, and moderately higher
blood pressure levels, compared with those individuals with the normal Met235 allele. In addition, the
Thr235 variant is associated with increased risk of preeclampsia during pregnancy.
We will employ the “mutagenically separated” PCR strategy to determine each student’s promoter
genotype from a blood sample. The promoter genotype is frequently associated with the coding
genotypes as listed in the first paragraph, but is not completely diagnostic. First, genomic DNA will be
isolated from the sample. Then, the sample will be subjected to two separate PCR reactions. In reaction
#1, we will use a reverse primer which matches the normal nucleotide; in reaction #2, we will use a
reverse primer which matches the SNP nucleotide. Both primers use the same forward primer so the
products are similar; however, the reverse primer used in reaction #2 is longer, so the PCR product
(amplicon) is larger for reaction #2 than in reaction #1. Following PCR (next week), our reactions will be
run on an agarose gel to determine which of the two alleles we have.
Protocol
1. Isolate DNA from your own blood sample using the miniprep protocol:
A. Obtain a blood sample
1. Wipe the side of the ring finger on your non-dominant hand with an alcohol wipe.
2. Use a lancet to create a small cut on your finger.
3. Collect ~200uL of blood in a 1.5mL centrifuge tube, which contains the anticoagulant
EDTA.
4. Put a band-aid on your finger, and label your tube. Mix it by inverting upside down
gently for 30 seconds. Place it on ice while you prepare the DNA extraction process
B. Extract DNA
1. Lyse blood cells
***If your blood sample is less than 200 uL, add PBS (phosphate-buffered
saline) to make it 200 uL. If you have between 200 and 300 uL of blood, use the
whole sample. If you have more than 300 uL, use ONLY 300 uL. In that case,
transfer 300 uL to a new tube***
BIO 330 Cell Biology
Laboratory
Spring 2011
a. Add 20uL of proteinase K to your blood sample
b. Add 400 uL Lysis buffer type 10 to your blood sample; vortex to mix.
c. Incubate 10 minutes at room temperature. Spin down briefly.
2. Bind genomic DNA to the column.
a. Assemble a column and collection tube; place in a rack.
b. Load your blood sample onto the column, then close the cap.
c. Centrifuge for 60 seconds at 11,000 x g.
d. Carefully discard flowthrough into glass beaker marked “WASTE”; put the
column back into the same collection tube.
3. Wash DNA
a. Add 500 uL Lysis buffer type 10 to column
b. Centrifuge 60 seconds at 11,000 x g.
c. Discard flowthrough again and replace column into same collection tube
4. Wash and dry DNA
a. Add 500 uL Wash buffer type 6 to column.
b. Centrifuge 3 minutes at 11,000 x g; discard flowthrough again, but DO NOT
REPLACE COLUMN IN SAME COLLECTION TUBE.
5. Elute DNA
a. Insert column into a clean DNase-free 1.5mL microcentrifuge tube
b. Add 200 uL Elution buffer type 5 (preheated to 70°C).
c. Incubate for 1 minute at room temperature.
d. Centrifuge for 60 seconds at 11,000 x g.
e. KEEP FLOWTHROUGH (i.e., collect the eluate, which contains your DNA).
Discard column.
f. Label the tube with your name and date, and the words “genomic DNA”.
2. Prepare two DNA reactions, one each in two 0.5mL PCR tubes:
Reaction 1:
Water
16
Reaction2:
PCR Master Mix
25
Forward primer
2
Reverse primerA
2
DNA sample
5
Total volume
50
Water
PCR Master Mix
Forward primer
Reverse primerG
DNA sample
Total volume
Add 30 uL mineral oil very carefully over the top of each of the reactions. The best way to do
this is to slowly pipette the mineral oil against the inside of the tube at the top, and let the oil slowly
flow down over the top of the reaction.
Label your tubes with your initials and the reaction number.
16
25
2
2
5
50
BIO 330 Cell Biology
Laboratory
Spring 2011
3. The PCR reactions will be run overnight in the PCR machine, then stored in the refrigerator until next
week’s lab session. At that time, you will perform electrophoresis to analyze your samples. The PCR
cycling protocol to be used is as follows:
94°C 5 min
40 cycles of:
94°C 60 sec
62°C 60 sec
72°C 60 sec
72°C 10 min
4°C
Hold indefinitely
4. DNA electrophoresis of your PCR reactions
A. Prepare a 2% agarose gel
1. Weigh out 1 g agarose powder, pour into 125-mL Erlenmeyer flask.
2. Add 50 mL deionized water, swirl to mix, and use a kimwipe to plug the opening of
the flask.
3. Microwave for 30-60 seconds. Watch closely during microwaving…if you see it begin
to boil, stop the microwave immediately. Swirl to ensure it is mixed.
B. Cast the gel
1. Assemble the gel casting apparatus according to Dr. DuPriest’s instructions.
2. Make sure comb is in place to form wells
3. Pour hot agarose to a depth of ~3 mm. Let sit ~30 min, or until hardened.
4. Carefully remove comb and ends of gel casting apparatus.
C. Load the gel
1. Pour enough 1X TBE running buffer into gel rig to fill reservoir(s) and cover the gel.
2. Carefully load PCR reactions into wells in gel. Be careful not to poke holes in the
bottom of the wells, or to pipette material outside of the wells.
D. Run the gel.
1. Put lid on gel rig and connect electrical leads to power supply (ensure everything is
red to red, black to black).
2. Set power to high and start running…you should see bubbles rising from the wire
near the wells.
3. Run for ~45 minutes.
Analyze your results
If you have a band in the lane with your “A” reaction, then you have an allele with the “A”
promoter variant, and you likely have the M235T SNP as well – these are the genotypes associated with
higher angiotensinogen protein level, higher blood pressure, and increased risk of preeclampsia in
BIO 330 Cell Biology
Laboratory
Spring 2011
pregnancy. If you have a band in the lane with your “G” reaction, then you have an allele with the “G”
promoter (normal) variant, and you likely have the normal M235 variant of the angiotensinogen protein.
If you have a band in both, you are heterozygous. If you have a band in only one or the other reaction,
you are homozygous for the allele showing the band in the gel.
Discussion
Discuss your results. Did you get what you expected? Discuss possible reasons your results
might be false positives or false negatives. Discuss what your results mean, in terms of health risk.