Technology & DNA

							                                                      Technology & DNA
                                                          Exercise #4
                                     Gel Electrophoresis of TPA-25 Amplification Products


Background: Now that you have extracted and amplified your TPA-25 locus, its time to see what your
genotype is. To do this, you will have to run a gel electrophoresis, loading the gel with the DNA that you
amplified. We will be using a special kind of gel, called a “Metaphor agarose” gel, which is very fragile, and
will be handled by your instructor. In addition, the gel will need to be stained after the run, since the bands
are not visible under normal conditions. The stain used, Sybr green, is highly toxic—so that will also be
handled by your instructor.

Equipment:
Electronic balance                                   Microwave oven
100 ml graduated cylinder                            500 ml Erlenmeyer flask
50 ml Erlenmeyer flask                               Electrophoresis apparatus
Power supply                                         Vortex mixer
Shortwave UV transiluminator                         Hooded Polaroid camera with orange filter or digital camera
100 l micropipettes (yellow tips)

Supplies:
Metaphor agarose                                     TAE buffer
Distilled water                                      Sybr green
Type 667 Polaroid film                               Micropipettes
Pasteur pipettes                                     Saran wrap
Purple sample buffer                                 50 ml centrifuge tube
Weigh boats

Procedure:
1.   Pour 4 1.8% (w/v) Metaphor agarose mini-gels: Using graduated cylinder, measure out 85 ml of freshly prepared 1X
     TAE buffer and pour into flask. Weigh Metaphor agarose (1.5 g.) and add to 500 ml flask containing buffer.
     Cover top of flask with Sarah wrap, punch a hole in wrap.
2.   Place flask on top-loading balance and tare weight to zero.
3.   Place 500 ml flask and 50 ml flask containing super water into microwave oven. Microwave on low setting for
     about 5 minutes, swirling from time to time. Repeat microwaving until agarose solution is clear and no “floaters”
     remain.
4.   Place flask on balance and using Pasteur pipette, add hot super water to readjust weight to zero (this
     compensates for evaporation).
5.   Assemble mini-gel with bumpers and one comb. Check orientation using lid and leads: the comb (origin) end
     should be at the end with black leads. Make sure it is level. Place 8-well comb at origin end of tray. The comb
     should be oriented so that its teeth are slightly away from the end of the tray, not up against the end (to leave
     room for the slots to form.
6.   When agarose has cooled to about 70o C (just hot to the touch), pour 20 ml into each tray (use 50 ml centrifuge
     tube to measure volume). Use Pasteur pipette to pop any bubbles.
7.   Leave gel undisturbed for at least half an hour—it will turn grayish. Pour TAE buffer to cover gel and place in
     refrigerator. Chill 1 liter bottle of TAE buffer in refrigerator at same time.
8.   Carefully and slowly pull comb out of gel, checking wells. (If any wells are damaged, these should be avoided if at
     all possible). Carefully remove bumpers. Rotate gel into running position (well = origin at negative (black)
     electrode). Fasten leads into power supply. Check orientation twice!
9.   Cover gel with TAE buffer, anchoring gel by placing one finger at the front end of the tray. (Metaphor gels are
     very slippery and may get pushed off the tray otherwise!) There should be just enough buffer to cover the entire
     gel about 1 mm (check from side).
10. If you are not yet ready to load samples, replace gel apparatus in refrigerator and chill until you are ready. (Gels
     can be kept overnight, or over a weekend)
11. At the end of the cycler run, press “stop” twice and turn off the cycler. Wipe off any condensation that has
     formed in the heating block. Carefully lift out tube rack. Check (and if necessary, replace) labels on tubes.
     Transfer tubes to external racks.
12.   Add 7.5 l of purple sample buffer to each tube, using 100 l pipette. Mix briefly with Vortex mixer, then pulse in
      microcentrifuge to mix phases. The sample buffer contains glycerol, which makes the sample dense.
13.   Using a 100 l pipette, load the samples in the following order, from left to right:
      Lane 1     10 l DNA ladder (already measured out)
      Remaining lanes:       12 l of each PCR reaction. Record order of samples on gel (Student initials). Store
                 remaining sample in freezer.
14.   Electrophorese for 1.5 hours at 70 v. Using casting tray, carefully slide each gel into a weigh boat containing Sybr
      Green. Stain for 15 minutes in dark (drawer or under foil) agitating gently from time to time. Note: staining will
      be more intense if you stain for 45 minutes. Careful, the gel is fragile.

                                                         Analysis

1.    Look at the DNA “ladder”—which is in Lane #1. The lowest and brightest band is at 123 bp, the next is at 246 bp,
      the next at 369 bp, and so forth, in other words, the bands are 123 bp apart. Now compare the DNA ladder with
      the amplified fragments of student DNA. Near the bottom of each lane, well below the position of the lowest
      band on the ladder, you will see a hazy band. This is known as the “primer dimer”, and is a byproduct of PCR
      reactions. It represents the unused primers, which stick together and copy each other. It is a “meaningless”
      band.

2.    Above the “primer dimer”, look at the bands in the next 7 lanes and compare their distances with the 123 bp
      ladder. Above the “primer dimer” band, you should be able to identify a 100 bp fragment band (the Alu-absent
      allele) and a 400 bp fragment (Alu-present allele).

3.    Look at the individual lanes, which correspond to student genotypes. Identify each as a homozygote or
      heterozygote, based on whether they have a single band or both bands. Identify homozygotes as Alu-absent or
      Alu-present based on which band is present. Place your information on the Table on the board at the front of
      the room.

4.    Make a table listing the genotype of each student in the class on this paper. (For a homozygous genotype, list 2
      “+” or 2 “-“, for a heterozygous genotype, enter a – in the appropriate column and a + in the appropriate column.)

                        Name of student                          Alu-absent (-)      Alu-present (+)      Genotype (+/+,
                                                                                                             +/-. -/-)
      1
      2
      3
      4
      5
      6
      7
      8
      9
      10
      11
      12
      13
      14
                             Totals

5.    Count the total number of Alu-present and Alu-absent alleles in the class. Calculate allele frequencies for Alu-
      present and Alu-absent (i.e., the number of each allele divided by the total number of alleles in the class.)

           Frequency of “+” allele

           Frequency of “-“ allele
     How do these calculated allele frequencies compare to populations in general? (In the U.S. population as a whole,
     Alu-present is about 60% and Alu-absent is about 40%. You should know that both alleles are found in all human
     populations. However, frequencies differ for different populations/ethnic groups.) Are they similar? Different?
     Explain why?




6.   Using the allele frequencies for the U.S. population as a whole, use the Hardy-Weinberg equation (p2 + 2pq + Q2
     =1) to predict the frequency of the three genotypes: heterozygous, homozygous (Alu-present) and homozygous
     (Alu-absent) in the U.S. Population.

          Frequency of +/+

          Frequency of +/-

          Frequency of -/-




7.   Using the allele frequencies you actually observed in class, calculate the predicted genotype frequencies for the
     class.

          Frequency of +/+

          Frequency of +/-

          Frequency of -/-



     Do these frequencies appear to be in Hardy-Weinberg equilibrium? If not, speculate on why they are not in
     Hardy-Weinberg equilibrium.