Lab Exercise 2: Using Allozymes to Identify Population Structure Protein gel electrophoresis is a technique that has been widely used to examine within and between population variation. Proteins are the phenotypic expression of DNA sequence and this variation reflects changes in protein-coding genes. In this procedure proteins are extracted and applied to a solid medium (starch, acylamide, cellulose acetate) and then separated based on charge. Charge differences are due mainly to changes in amino acids lysine, arginine and histidine (basic or negative amino acids) and aspartic acid and glutamic acid (positively charged amino acids) and reflect changes in amino acid sequences due to mutations in the encoding DNA leading to charge differences. The alleles are visualized by histochemical staining which consists of an enzyme-specific substrate, the necessary cofactor and oxidized salt that links the enzyme and results in a dye precipitate. The number and types of alleles at each locus are recorded and used to examine population structure through the use of a series of population genetic parameters. The genotypes are recorded (i.e, AA, Aa, aa) and the allele frequencies are determined as:
 
p = (2NAA + NAa)/2N
q = (2Naa + Naa)/2N


Observed frequencies are then tested for agreement with the Hardy-Weinberg principle.

There are many advantages to this technique over other, more recently developed molecular techniques. Advantages include the fact that this procedure is relatively inexpensive, easy to conduct, quick and one is working with codominiant markers where homozygotes and heterozygotes can be distinguished. Disadvantages to the technique include the fact that one is only surveying a small fraction of the genome and this region tends to be a more conserved (i.e. coding) region. Finally, although generally polymorphic enough for most studies, allozyme loci aren't as polymorphic as some molecular markers such as SSR.

In this lab we will examine the structure of a population of mosquitoes (Adedes aegypti). The original collection of several hundred mosquitoes was made in West Timor, Indonesia. The mosquito population that we will be analyzing is an F2 population that has been maintained in the lab. We will screen a subsample of this population for polymorphic loci using protein gel electrophoresis (allozymes). We will then use the program Genepop3 (Raymond and Rousset 1995) to examine the structure of this population. You will first calculate allele frequencies, and then test to see if this population is in Hardy-Weinberg Equilibrium (probability test) and if not, test for heterozygote deficiency or excess.

Procedure:

I.    Gel and gel tank preparation:

           1.     Label the back of each plate (mylar side) with the enzyme to be used.

 2.    Soak cellulose acetate plates in the same buffer as the electrode (running) buffer. In our lab this will be the Tris-Glycine buffer. When the plate is immersed in the buffer, care must be taken to avoid bubble formation on the plate as it is immersed. To do this, we place the gels in the soaking chamber and then let the buffer slowly flow in from the chamber above. Gels must soak for at least 20 minutes prior to use.

3.    Set up the gel tank by placing electrode (running) buffer in each section and placing wicks along the support rails. The buffer level should be up to the level of the wicks and even across each reservoir.
 

II. Sample preparation: Samples that are collected and not run immediately should be frozen in liquid nitrogen and stored at -70o until needed. When you are preparing your sample for allozyme analysis, it is important to keep the sample frozen (we'll process our samples on dry ice) until the sample is in the grinding buffer.
1.     On dry ice, remove a single mosquito and place into a chilled, labeled eppie, on dry ice. Use chilled tools (forceps, etc.) to handle the frozen sample to prevent thawing.

2.    or Bloodroot - crush the leaf tissue in the tube, on dry ice using a chilled pipet tip, then proceed to #3

3.       Doing one sample at a time (and keeping the rest on dry ice), add 20 ul of Tris-glycine grinding buffer to the sample, break up the tissue using a pipet tip or dounce, and store on ice. (Use 120 ul Tris-glycine +PVP grinding buffer for bloodroot). Repeat for each sample. Spin each sample for 15-30 seconds to pellet tissue in the bottom of the tube and store on ice.
 

III. Sample loading:
 
1.   Record gel loading order and then place the sample plate on ice and add 10 ul of extract to each well in the sample loading order.

2.    Remove a gel plate from the soaking buffer and blot dry between two piece of paper toweling. It is important to blot the gel dry as excess buffer on the gel surface will cause the sample to spread and result in broad bands that will be hard to resolve.

3.   Place the plate on the aligning base, mylar side down. To prevent the plate from moving, you can apply to few drops of running buffer to the aligning base to help hold the gel in place. Position the plates so that there is space at the top (~3-4mm) so that the plate can sit on the support rails without coming in contact with the samples.

4.   Please use extreme care when using the applicator as it as very expensive and the metal loops are easily bent and broken. Always be sure that it is aligned with the aligning base or sample wells before applying pressure. Never force the applicator.

5.   Place the applicator in position to be sure that the plate is aligned correctly so that all samples will be applied. (Note: we will run either 9 or 12 samples, depending on which plates we are using).

6.   Once the applicator and plate are aligned, transfer the applicator to the well plate and load the applicator.

7.   Transfer applicator to the aligning base and apply the sample to the gel.

8.   Apply the sample to three plates. This is the number that will fit on the gel tank and we will use three different stains to detect allozyme variation.

9.   Once you load a plate, place it on the wicks in the tanks (without current), while you are loading the rest of the plates. This will keep the plates from drying out. Place plates acetate side down. Be sure that the zone that contains the samples does not come in contact with the wicks.

10.   If you are using different samples, the applicator must be cleaned by blotting its teeth on filter paper before using another extract.

III. Gel Running:
 
1.     The current runs from the negative (cathodal) to positive (anodal) electrode and the load zone should be positioned at the negative side for the majority of enzymes systems which migrate from negative to positive. (Note: for those systems which migrate in the opposite direction [positive to negative], the extracts should be loaded near the center of the gel).

2.     Make sure that there is no air space between the gel and the wicks.

3.   Run the gel at 200 volts for 15 min. While the gel is running, start preparing the stains.

     
IV. Staining:
 
1.   While the gel is running you should start preparing the stains. Many of the components can be mixed ahead of time.

2.   Make up the stains by putting the appropriate reagents into a scintillation vial. Mix the first set of reagents while the gel is running and then add those marked with an **

    3. To stain: Turn off the current and add the final reagents to the stain.

    4.   Stain one plate at a time by removing one plate at a time from the tank and leaving the rest on the support rail with the current off.

    5.   Place the plate in a plastic tray, being sure that the gel lies flat and does not dry out.

    6.   Add the agar to the stain, mix well by swirling and then pour the stain on the center of the plate

    7.   Let sit for one minute to allow the agar to harden.

Stains:
 
Glucose-6-Phosphate Isomerase (GPI) Phosphoglucomutase (PGM)
1.0 ml Tris-HCl, pH= 8.0 1.0 ml Tris-HCl, pH= 8.0
1.5 ml NAD 1.5 ml NAD 
5 drops Fructose-6-phosphate  5 drops MgCl2
5 drops MTT  5 drops Glucose-1-phosphate soln
**5 drops PMS 5 drops MTT
** 10 ul G6PDH **5 drops PMS
2 ml agar  ** 20 ul G6PDH
  2 ml agar
4o structure - dimer 4o structure - monomer

** Add immediately before staining (i.e., after the gel is finished)
 
Malate Dehydrogenase MDH (#21) Isocitrate Dehydrogenase IDH (#18)
1.0 ml Tris-HCl, pH= 8.0 1.0 ml Tris-HCl, pH= 7.0
1.5 ml NAD 1.5 ml NADP 
13 drops Malic substrate 15 drops DL-Isocittrate
5 drops MTT 8 drops MgCl2
**5 drops PMS 5 drops MTT
** 10 ul G6PDH **5 drops PMS
2 ml agar ** 10 ul G6PDH
  2 ml agar
4o structure - dimer 4o structure - dimer, usually
# of isozymes: 2; cathodal band is mitochondrial form; anodal band, supernatant form  # of isozymes: 2, faster zone is subject to breakdown; slower mitochondrial form stains intensely

** add immediately before staining (i.e., after the gel is finished)
 
 
 
Mannose-6-Phosphate Isomerase MPI (#23) Asporate Amiro Transferase AAT 
1.0 ml Tris-HCl, pH= 8.0  3 ml Solution # 1
1.5 ml NAD drops Fast Blue BB salt (saturated
5 drops D-Mannose-6-Phosphate Solution or add a small amt to solution #1)
5 drops MTT  2 ml agar
**5 drops PMS  
**5 ul PGI Solution #1
** 20 ul G6PDH 200 ml 0.1M Na Phosphate, pH =7.0
2 ml agar  10 mg Pyridoxal -5-phosphate
4o structure - monomer 40 mg L-Aspartic acid
  260 mg a-Ketoglutaric acid
  Adjust to pH=7.4 This step is critical!
  4o structure - dimer
  # of isozymes - 2; anodal zone corresponds to the supernatant form; weakly cathodal zone to the mitochondrial form

** add  immediately before staining (i.e., after the gel is finished)