DNA BARCODING PAPER NEEDED FOR TOMORROW
DNA BARCODING OF BEES: Introduction: Every animal is important to our ecosystem, but one of the most significant animals are the bees, specifically the honeybees. Bees are insects that provide high-quality food and other products used in healthcare and other sectors. The most important contribution of bees is the pollination. The pollination of the plants produce 90% of the world’s food. That means a third of the world food depends on bees. The truth is that bees are among the most important pollinators in the world, this means that in order for a plant to reproduce it needs a pollinator to transfer its pollen from one flower to another so that the plants get fertilize and can produce a new species (Williams et al., 2015). If bees start becoming extinct their abscesses can tell us when something is happening, and we need to take action. According to the report, the bee population has decreased by 40.7% (Watanabe., 1994) In the state of Florida live 315 species of native bees, of which 29 are endemic, which means only found here. These 315 species can be found in six different families including Halictidae, Megachilidae, Andrenidae, Apidae, Colletidae and Melittidae. Differential impacts from an extreme cold event on a native, subtropical bee vs. an introduced, tropical bee can also affect native bees and their habitat. (Downing & Borrero & Liu., 2016). DNA Barcoding is the process to identify every species using an example of DNA from that species gene or genes. It constitutes a very accurate and credible process for the species identification. The results also exposed some surprises, counting with the identification of two unique species, one in the genus Ceratina and the other in the genus Andrena. Method: DNA extraction: DNA extraction was performed. First, the bees were cut in half and one of the sides was smashed. Detergent and sodium ions were added to break down lipids and to protect the negative phosphate groups, proteins needed to be removed. This was accomplished by using a protease, finally we filtered and purified with Zymo-spin IIC Fast-spin column. 1.Add specimen to a ZR BashingBead Lysis Tube (2.0 mm). Add 750 μL BashingBead Buffer to tube, put cap on tightly
2.Secure ZR BashingBead Lysis Tube in a bead beater (Disruptor Genie) and process at highest speed for 10 minute
3.Put ZR BashingBead Lysis Tube in a microcentrifuge and spin at ≥ 10,000 x g for 1 minute
This will separate out sample into solid precipitate and liquid supernatant
4.Transfer 400 μL (or less) of the resulting liquid from Step 3 to a Zymo-Spin III-F Filter in a Collection Tube, centrifuge at 8,000 xg for 1 minute
The liquid that passes through the filter is now called a filtrate
5.Add 1,200 μL of Genomic Lysis Buffer to the filtrate in the Collection Tube from Step 4 and mix 6.Transfer 800 μL of the mixture from Step 5 to a Zymo-Spin IIC Column in a Collection Tube and centrifuge at 10,000 x g for 1 minute 7.Discard the flow through from the Collection Tube and repeat Step 6 8.Add 200 μl DNA Pre-Wash Buffer to the Zymo-Spin IIC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute 9.Add 500 μl g-DNA Wash Buffer to the Zymo-Spin IIC Column and centrifuge at 10,000 x g for 1 minute. Perform dry centrifugation. Keep tubes on ice if needed
10.Transfer the Zymo-Spin IIC Column to a clean 1.5 ml microcentrifuge tube and add 100 μl DNA Elution Buffer directly to the column matrix. Incubate for 1 min at room temperature. Centrifuge at 10,000 x g for 1 min to elute the DNA
11.Take the eluted DNA back to the Zymo-Spin IIC Column and centrifuge at 10,000 x g for 1 min to elute the DNA to re-elute the DNA. Perform dry centrifugation at 10,000 x g for 1 min
Clean and Concentration: 1.When the DNA is successfully bound to the silica, there is still residual protein that needs to be washed off 2.Ethanol is usually added to a wash buffer and when centrifuged, the DNA stays within the silica and everything else is washed away
Use DNA Clean & Concentrator-5
1.Add 24 mL of 100% ethanol to 6 mL of DNA Wash Buffer
2. Add 200 μL 2.DNA Binding Buffer to your DNA sample, mix briefly by vortexing
3.Transfer mixture to a 3.Zymo-Spin Column in a Collection Tube
4.Centrifuge at ≥10,000 x g for 30 seconds. Discard the flow-through
5.Add 200 μl Wash Buffer to the column. Centrifuge at ≥10,000 x g for 30 seconds. Repeat wash step 6.Add 20 μl of Elution Buffer directly to the column matrix. Transfer the column to a 1.5 ml microcentrifuge tube and centrifuge at ≥10,000 x g for 30 seconds to elute the DNA
Ultra-pure DNA in water is now ready for use!
“Pure” DNA will have a 260/280 ratio of around 1.8 The DNA is heated to denature it, then, it is cold down for the primer to bind to a specific portion to make a starting point for the copying process to take place. In this case, the gene amplified was COX1, which is important because it exhibits small changes between organisms of the same species. A step called elongation occurs last, in which the DNA is heated again and taq. polymerase starts producing copies of the original strand using the nucleotide bases.
PCR- Denaturing – heating DNA up causes the double-strand to separate into 2 single strands of DNA
Annealing – temperature is lowered so DNA primers attach to the single strands template
◦ Primers mark the target gene, Taq polymerase enzyme binds to primer Extending – temperature is raised again, Taq polymerase makes a new strand of DNA by attaching DNA bases to the single strand
Gel Electrophoresis-
1. Put the sample inside the wells 2. Do not break the sample gell. 3. Make sure you do not release the top bottom of the pipette. Otherwise the sample will
come back to the pipette. Running the Gel:
1. In the Gel box we need to place the agarose gel 2. To cover the gel, we need to put enough TAE saline buffer inside the gel box 3. Hook gel box up to battery 4. Make sure to put the negative side in the wells side 5. Pipette carefully DNA Ladder and DNA sample 10uL inside the wells 6. observe and record which sample get into which well 7. Battery need to be run for about 45 minutes to be able to see separation 8. Take pictures of the bands with UV light
References: Downing, J., Borrero, H., & Liu, H. (2016). Differential impacts from an extreme cold spell on subtropical vs. tropical specialist bees in southern Florida. Ecosphere, 7(5). Williams, N. M., Ward, K. L., Pope, N., Isaacs, R., Wilson, J., May, E. A., ... & Peters, J. (2015). Native wildflower plantings support wild bee abundance and diversity in agricultural landscapes across the United States. Ecological Applications, 25(8), 2119-2131. Elzen, P. J., & Westervelt, D. (2002). Detection of coumaphos resistance in Varroa destructor in Florida. American Bee Journal, 142(4), 291-292. Watanabe, M. E. (1994). Pollination worries rise as honey bees decline. Science, 265(5176), 1170-1171. Cartwright, B. A., & Collett, T. S. (1983). Landmark learning in bees. Journal of comparative physiology, 151(4), 521-543.