As we discussed in class, the Hardy-Weinberg equation can be used to predict allelic and genotypic frequencies within a population. The Hardy-Weinberg equation, p2 + 2pq + q2 = 1, states that p2 reresents that number of homozygous (+/+) individuals, q2 represents the number of homozygous (-/-) individuals, and 2pq represents the number of heterozygous (+/-) individuals within a population. Of course, H-W equilibrium only occurs if the population is large, mating is random, and none of the genotypes confer a selective advantage.
To investigate H-W equilibrium, we will determine our genotypes for PV92 locus of chromosome 16. PV92 is a non-coding locus; as a result, no selective pressure should influence genotypic frequencies. Furthermore, because PV92 is a non-coding region, mating with respect to this locus should be random. Finally, PV92 is dimorphic; that is, two alleles exist within the human population. One allele contains a 300 basepair Alu insertion, while the other allele does not contain this insertion. Not surprisingly, then, the two alleles can be differentiated by PCR amplification of the PV92 locus and agarose gel electrophoresis separation of the PCR products. We will use these techniques to determine the allelic and genotypic frequencies of PV92 in the members of our class and determine if we are in H-W equilibrium.
Day 1: DNA Isolation and PCR Amplification
Using a pipet tip, scrape the inside of your cheek approximatley
ten times and place the tip inside a screwcap tube containing 200 ul of InstaGene
Repeat with a second tube of InstaGene matrix.
For each tube, place the tip on a pipettor and pipet up and down five times.
Screw on the tops and incubate at 56oC for 10 minutes. NOTE: After 5 minutes, vortex the tubes and then return to the water bath.
Remove the tubes, vortex, and incubate at 100oC for 6 minutes.
Remove the tubes and vortex. Then, pellet the matrix by spinning the tubes for 5 minutes at 6,000 xg.
Remove 170ul of supernatant from tube 2 and add it to tube 1. Repellet the matrix by spinning this tube for 2 minutes at 6,000 xg.
Transfer 20ul of your DNA solution to a 0.5mL tube. Be careful not to transfer any of hte matrix beads.
To this tube, add 20ul of the PCR master mix. Mix by pipetting up and down several times.
Place your tube in the thermal cycler.
After the PCR reactions have been completed, I will transfer the tubes to a rack in the freezer.
Each group must pour a 1% agarose gel in 1x TAE. Store your gel in 1x TAE until you are ready to run your samples.
1) The InstaGene matrix consists of negatively charged beads that serve as metal ion chelators. Many enzymes, such as DNases, require metal ions as necessary cofactors. By chelating these cations, then, we are inactivating these enzymes. As a result, we are protecting our genomic DNA from degradation by cellular enzymes that have been released during the DNA isolation protocol.
2) The incubation at 56oC serves two purposes. First, it helps to break apart clumps of cells. Second, it helps to inactivate DNases that will be present in the mixture. The incubation at 100oC ruptures the cell membranes, thereby releasing the genomic DNA.
3) Master Mix: 2x mixture of PV92 primers, Taq DNA polymerase, nucleotides, and buffer.
4) PCR Program:
1. 94 C for 2 minutes
2. 94 C for 1 minute
3. 60 C for 1 minute
4. 72 C for 2 minutes
5. Repeat 1. through 4. 40 times
6. 72 C for 10 minutes
Day 2: Gel Electrophoresis and Data Analysis
Add 10ul of 5x loading dye to your PCR tubes.
To your gel, load 20ul of each of your samples. Also, load 20ul of the homozygous (+/+) control, 20ul of the homozygous (-/-) control, and 20ul of the heterozygous (+/-) control. Finally, add 10ul of the MW marker.
Run the gel at 100V for 30 minutes.
Remove the gel from the electrophoresis apparatus and place it in a container of ethidium bromide for 15 minutes.
Observe under UV light and take a picture of the gel.
Record results on the board.
1) Ethidium bromide is a suspected carcinogen! Always wear gloves when working with EtBr.
2) The MW marker contains DNA fragments of 100, 200, 500, 700, and 1000 basepairs.
3) The PV92 locus without the Alu insert should result in a PCR product of 641 basepairs. The PV92 locus with the Alu insert should result in a PCR product of 941 basepairs.