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Extract DNA

Amplify DNA

GENESCAN

Analysis

In order to identify the microsatellites on the Pacu fish, the following protocol had to be followed. 

Step 1:  Extracting the DNA from the nucleated blood cells of Pacu fish.

The procedure will follow the steps of the DNA extraction technique created by the Qiagen DNeasy Kit.

1. The samples were obtained by Daniela Calcagnotto's research in Brazil, South America.  Tubes of blood cells from different fish of the same species were stored in an ethanol solution.  To extract the DNA, a very small amount of blood was taken with toothpicks and placed in the bottom of a sterile Eppendorf tube.
2. Place tubes containing blood sample in an incubator at 65^oC for 15 minutes to evaporate off the ethanol in which it was stored.
3. Add 190 microliters ATL (Tissue Lysis Buffer) buffer and 10 microliters of   Proteinase K to the blood tissue.   Incubate at 65^oC for 10 minutes.  Take tissues out of incubator and vortex to mix.  Continue incubate/vortex cycle until cells are digested which should take approximately 30 minutes. 
4. Add 200 microliters of AL buffer, vortex and incubate at 70^oC for 10 minutes.
5. Add 200 microliters ETOH and vortex.  Pipet mixture into spin column with collection tube.  Centrifuge at 8000 for 1 minute.
6. Transfer the column to a fresh collection tube and discard the initial collection tube, and add 500 microliters of AW2.  Centrifuge for 3 minutes at full speed.
7. Place the column in a fresh 1.5 ml Eppendorf tube and pipet 200 microliters of AE buffer to wash the DNA off the column.  Incubate at room temperature for 1 minute, then centrifuge for 1 minute. 
8. Store the DNA in the refrigerator until the sample is needed.  The DNA can also be stored in the freezer is necessary.

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Step 2:  Amplifying the DNA.

To identify four different microsatellite loci, the extracted DNA from the nucleated fish blood was mixed with four different primers and amplified with the a PCR machine (PCR Demonstration).

Using the tubes from the above DNA extraction procedure, the DNA was mixed with a different primer for each amplification.  The primers used were Pme4, forward and reverse; Pme21, forward and reverse; Pme14, forward and reverse; and Pme2, forward and reverse. 

Procedure used for primer Pme4 and Pme14.

1. Using PCR tubes with beads that contain Taq, dntp and buffers, the following materials were placed in each tube in the order given. 

• 23.5 µl of H₂0
• .5 µl of primer (i.e. Pme4 forward)
• .5 µl of primer (i.e. Pme4 reverse)
• .5 µl of DNA sample

If no PCR tubes with beads are available the following master mix may be substituted to create a final volume of 25 microliters.

• 2.5 µl of buffer
• 2.5 µl dNTP
• .5 µl of primer (forward)
• .5 µl of primer (reverse)
• 2.5 µl of BSA
• .1 µl of Taq
• 15.9 µl of H₂O
• .5 µl of extracted DNA

2. A negative control, a sample containing no DNA, was also prepared for amplification to check for any contamination.  The negative control was prepared with the materials listed below following the order given. 

• 23.5 µl of H₂O
• .5 µl of forward primer
• .5 µl of reverse primer

3. After the tubes are mixed, all tubes containing above ingredients including negative control were place in a thermal cycler or PCR machine for amplification. 
4. The PCR program used decreased the annealing temperature from 59^oC to 51^oC in the first five cycles.  Once the annealing temperature reached 51^oC, the annealing temperature remained constant for the last 25 cycles.  The cycle sequence is listed below.  Primer Pme4 and Pme14 were run with the program cycles listed below.

• Denature temperature was set at 95^oC for 10 minutes

• Extend temperature was 72^oC for 10 minutes, then temperature drops to 4^oC.  Keep the PCR samples at 4^oC until ready to use.  

5. After PCR amplification is done, the samples were loaded on 1 % agarose gels.  The gels were made by mixing .8 g agarose with 80 ml of TBE buffer.  Heat mixture in microwave for 1 minute.  Remove from microwave, swirl.  Heat in microwave for another 30 seconds, cool in a water bath at room temperature.  Add .9 microliter ethidium bromide to the mixture and pour.
6. The wells were filled with 5 microliter of PCR product from above and 1 microliter of staining gel.  A negative sample was also loaded into a well to be used for comparison.  Run gels for 15 minutes at 150 volts. 
7. A new primer was used to identify the second loci on the different fish from the same species.  The following protocol was used with primer Pme21. 
8. A master mix was made containing scaled up volumes to equal the following ratio:

• 23.5 µl of H₂0
• .5 µl of Pme21 (forward)
• .5 µl of Pme 21 (reverse)
• .5 µl of extracted DNA from Step 1

24.5 microliters of master mix and .5 microliters of DNA sample was added to a clean PCR tube with bead.  A master mix was made by adding all of the above ingredients except the extracted DNA to the PCR tube.

9. The tubes were placed in a PCR machine using the following program.

• 95^oC for 10 minutes
• 94^oC for 45 seconds
• 59^oC for 45 seconds
• 72^oC for 1 minute
• Repeat for a total of 30 cycles
• Extend at 72^oC for 10 minutes.  Store at 4^oC until needed.
• Run gels with DNA PCR product and negative control. (See Step 6)

10. Using the master mix listed above for the 25 microliter volume, the last primer used was Pme2.  Place 24.5 microliters of master mix plus .5 microliters of DNA into PCR tube.  The following cycle sequence was used for primer2.

• 95^oC for 10 minutes
• 96^oC for 20 seconds
• 57^oC for 45 seconds
• 72^oC for 45 seconds
• Repeat for a total of 30 cycles
• 72^oC for 10 minutes.  Store at 4^oC until needed.
• Run gels with negative control (See Step 6)

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Step 3:  GENESCAN

Three gels were run on the PCR products from above.  The alleles were genotyped using GENESCAN 2.1 version and GENOTYPER 2.1 version softwares from ABI.   Our PCR sample from Pme4 and Pme21 were combined on the first gel.  The fluorescent dye that identified the two different primers was yellow, but the fragment sized produced should vary.  Since the primers 2 and 14 were labeled with the same fluorescent dye, blue; and the loci were of similar fragment size, individual gene scans were run on the PCR product.

• Formula used for Pme4 and Pme21
• 5 µl of H₂O (optional if you don't want to dilute DNA samples)
• 5 µl of PCR sample with Pme4 primer
• 5 µl of PCR sample with Pme21 primer
• Vortex mixed PCR samples

After H₂0 and PCR samples were mixed, new Eppendorf tubes were used to mix formamide and TAMRA, which was the size standard; and the DNA mixture. In the clean tube add the following ingredients in the order given. 

• 2.6 µl of formamide/TAMRA mix (TAMRA is our size standard)
• 1.5 µl of mixed DNA
• Vortex
• Place in refrigerator until gene scan gel (polyacrylamide) is ready.
• When gel is ready, place mixed PCR samples in 95^oC heat block for 3 minutes.  Then place on ice for 3 minutes.
• Polyacrylamide gel is loaded by placing DNA/formamide/TAMRA mix in wells that are odd number.  After all odd-number wells are loaded, run the gel for 2 minutes.  This allows leakage across lanes to be detected.  Then stop gene scan machine and load even number wells of gel. 
• Run GENESCAN computer software  in a gene sequencing machine. 

The results of GENESCAN will give us the fragment size of the different loci and identify whether the loci are homozygous or heterozygous.  Since microsatellites are noncoding repeat sequences of DNA, no dominant or recessive alleles are present.  Below are diagrams of the results of the gene scan.

Heterozygous GENESCAN

  GENESCAN with Primers

After our group charted whether the different samples were heterozygous or homozygous and identified the size of the respective loci, we made a worksheet on Excel comparing the fish sample number and fragment size(s) depending on whether loci were homozygous or heterozygous. 

Step 4:  ANALYSIS

Then using Daniela's research, depending on the primer, we converted the fragment size to the number of repeats that would make up the microsatellites.  Finally our results were statistically analyzed using the programs GENE POP and RSTCalc to determine whether there is any population substructuring among the species from the five different rivers. We analyzed the data using both Fst and Rst measures of genetic distance, which use the infinite alleles model of microsatellite mutation and the stepwise mutation model, respectively.  We also determined whether our loci were in Hardy-Weinburg equilibrium, and whether there was any linkage between the loci.

Resources:
Genepop on the Web
Information about RstCalc,
 

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