Objectives:
Polymerase Chain Reaction (PCR) is a fairly new method that won Dr. Kary Mullis the Nobel Prize. The method allow scientists to amplify a specific piece of DNA from a mixture of DNA. Since scientists can now have a large amount of a specific piece of DNA quite rapidly (as you will find in the experiment), they can do very precise analysis of the DNA from a variety samples. PCR has made great impact on several areas of biology such as population genetics, taxonomy, ecology, as well as molecular biology and genetics. Medicine and criminology have also been greatly affected by the technology. You can test for genetic disease or DNA fingerprint a spot of blood (like from the handle of a Ford Bronco). Since we have gone over the theory behind PCR, I will discuss the theory behind the DNA preparation and what DNA is amplified in the experiment.
The DNA preparation will be prepared from cheek cells isolated in a non-invasive procedure (spitting). Briefly, you will swish about 10 ml of saline (0.9% NaCl) around in your mouth for about 10-15 seconds. This will scrape off enough cells to do the DNA preparation and PCR about 100 times ( they have used this to identify mail bombers by the DNA on the back of a stamp they licked). We will collect the cells by centrifugation and resuspend the cells with resin call Chelex that will bind inhibitors of the PCR. The cells are then lysed by boiling and the cell debris is collected by centrifugation. The supernate contains the DNA ready for PCR.
The DNA that will be amplified is the locus of the human TPA-25 locus of chromosome 8. In the figure below, you can see two primers (Forward and Reverse) that flank the TPA-25 locus. The distance between the two primers is about 100 bp. In some humans genomes, there is a Alu insertion sequence between the two primer sites that increases the distance between the two primers to 400 bp.
By doing PCR on the DNA isolated from your cheek cells followed by agarose gel electrophoresis analysis, we can determine your genotype at this locus. I you only have a 400 bp band, you would be homozygous for the Alu insert in TPA 25. If you had one band of each, you would be heterozygous or one copy of TPA 25 had an insert of Alu and the other does not. If you have one band at 100 bp, neither copy of TPA25 contains an Alu insert. We will then use class data to work on population genetics (using the website at the end of the methods) and discuss ethical considerations of DNA typing.
Lab Session : DNA preparation
Supplies
| 1 | 0.9 % saline tube (10 ml) |
| 1 | 1.5 ml tube with 500 ul of 10% Chelex in 50 mM Tris pH 10.5 |
| 1 | Pasteur pipet w/ bulb |
| 1 | 1.5 ml microcentrifuge tube |
| 1 | PCR easy start tube |
| 1 | Tube of Molecular Biology Grade Water |
| 1 | Tube of SAD #1 (forward primer) per group |
| 1 | Tube of SAD #2 (reverse primer) per group |
| 1 | tube of Taq Polymerase for the class |
Equipment
Methods:
DNA preparation from cheek cells
1. Take 1 tube of saline, write down the number, This will be an anonymous DNA test so write down your number if you want to know your genotype at the end of the experiment.
2. Swish the saline around in your mouth for 10-15 seconds and spit into a cup. Pour the contents of the cup into the tube you had with the saline and bring the tube back up to the front of the lab for centrifugation.
3. After a 10 minute centrifugation, pour the supernate into the orange bag and pipet Chelex mixture into the large tube with Pasteur pipet. Pipet up the cells and down with the Pasteur pipet and finally pipet the entire contents of the tube back into the tube that you had the Chelex mix in the first place.
4. Bring the Chelex/ cell mix up to the front of the classroom and we will boil the cells for 10 minutes.
5. After boiling, the cells will be iced for one minute and then centrifuged in the microfuge for 1 minute.
6. Now pipet 100 ul with one of the micropipettors you used in previous classes into a clean microfuge tube and label it with your number.
Set up of PCR reactions
1. Dr. Dan has added the following to the PCR tube
2. Add 5 ul of your DNA to the solution on top of the wax plug in the tube.
3. Bring to the thermocycler on your side of the laboratory.
4. We will run 35 cycles of the following temperature profile:
4. When the PCR is done, we will analyze the results by Agarose Gel Electrophoresis.
Virtually all molecular biology experiments involving DNA
use agarose gel electrophoresis as a basic tool. Basically, DNA
in negatively charged because of the phosphate part of the sugar
phosphate backbone. If you put DNA in solution and apply an electric
field, the DNA will run to the positive pole. To separated DNAs
that are different lengths, agarose is used to set up a molecular
sieve. The DNA molecules try to find their way through this sieve.
The smaller molecules of DNA find their way through the sieve
faster that the large DNA molecules. Therefore, the smaller DNA
molecules move through the agarose gel faster and move further
down the gel as compared to the larger DNA molecules. We can use
this to analyze the size of each fragment of DNA.
Two things affect the agarose gel electrophoresis: the percentage
of agarose and the voltage applied during the electrophoresis.
The percentage of agarose affects the sieving of the DNA molecules.
If you are separating large fragments of DNA (10,000 bp or higher)
you will use a very low percentage gel (0.7% w/v or less). The
holes in the agarose will be larger and allow the DNA to find
its way through. If you were separating small fragments of DNA
(500bp or less) you want smaller holes in the agarose (to be a
better sieve). Here you would use 2% agarose. The voltage is based
on the size of the gel unit and the size of the DNA. If you have
a large unit, the distance between the poles will be larger and
you can have a higher voltage. The rule of thumb is no more than
5v/cm (the distance between the two poles. The higher the molecular
weight, the less volts per cm is better (so you give the DNA time
to find the holes in the agarose).
The setup of an agarose gel electrophoresis is fairly simple.
If you can make Jell-O, you can make an agarose gel for electrophoresis.
Simply, determine how large of DNA fragments you are separating
(the larger the DNA the less agarose you use, the smaller the
DNA the more agarose you use). Then, add the amount of agarose
to the volume of 1 X electrophoresis buffer. Heat until boiling
in the microwave and check that all the agarose has gone into
solution and add the ethidium bromide (the stain for nucleic acids).
Then pour the liquid agarose/buffer solution into the form and
put in the comb (to give us a place to put our DNA samples). We
then let that solidify and take out the comb. The comb depressions
are now the wells where we can put our DNA samples. We then put
the gel in the electrophoresis unit and add 1X electrophoresis
buffer to cover the gel. Then, the samples are mixed with loading
buffer (to help with your loading of sample and follow the electrophoresis).
Finally, we load the samples and standard DNA. Always load a standard
DNA sample to make sure the gel works correctly and it will help
us with the analysis. When all samples and standard are loaded,
connect the electric leads to the power supply and electrophoresis
unit. You are now ready to turn on the electricity and watch the
electrophoresis.
1. Pipette out 20 ul of your PCR sample. This may be difficult with the wax covering your sample. You have to stab through the wax to the sample. If the tip gets plugged wax , pop off the tip and get a new one. Then with that pipet tip, go through the hole you made before and pipette up the 20 ul of your sample.
2. Mix your sample with the electrophoresis loading buffer on
the parafilm by pipetting up and down.
3. Carefully pipette the sample mix into one of the wells in
the agarose gel. Make sure you write down which wells you have
your samples and standards in.
4. When all samples and standard have been loaded, place lid on top of electrophoresis unit with red to red and black to black. Connect leads to the power supply and start power supply. We will run this gel at 100-125 volts.
5. Analyze by taking a picture of the gel using UV light box.
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