The Search for the Emerald Ash Borer Genomic Fingerprint by means of PCR and Gel Electrophoresis.

By: Marisa Solano-Sanchez

LBS 145

Section Th1

Dr. Luckie

April 28, 2005

Abstract

The Emerald Ash Borer (EAB) is a major threat to the vulnerable ash trees that are in Michigan . With complete identification, the threat can be recognized early on and save further infestation. This experiment takes on finding a replicable genome fingerprint of the EAB. The identification of this pest at a genetic level can be used in future infestations. Three main steps were taken to attempt the genome fingerprint, extraction of DNA, amplification of DNA using PCR, and gel electrophoresis. Extraction of the EAB DNA was from a mature alcohol preserved larva using a confirmed protocol. When DNA presence was confirmed, Polymerase Chain Reaction (PCR) was used to amplify the DNA using pre-existing DNA primers. After PCR produced an ample amount of DNA to work with, gel electrophoresis was the last step. The gel electrophoresis uses an agarose gel as the medium thru which an electric current was administered. The DNA, lambda ladder and positive controls were put in the wells of the gel tray and were dyed for better visualization. Bands of DNA are apparent under ultraviolet light if the primers bind correctly during PCR and create amplified DNA across the gel. In this experiment, the result of these procedures did not yield any bands of DNA across the gel tray, therefore a replicable genome fingerprint was not found.

Figure 3: Confirmation of DNA in Eppendorf tube one.

After the unsuccessful trial one of PCR, there was doubt as to whether or not DNA was present. This is evidence of DNA, as the ethidium bromide is binding to the DNA and produces flouresence. The negative control was distilled water; positive control was potato DNA, and tube one was done twice to ensure correctness. This test was done on all three Eppendorf tubes containing the EAB DNA. All of the tubes were confirmed to have DNA.

  Discussion

The Emerald Ash Borer was chosen to be studied because of its large impact on the environment that Michigan forests are facing. An example of a successful study involves our neighbors to the north and another foreign insect that inhabits susceptible forests. The Asian Longhorn beetle was also recently discovered and has threatened all of Canada 's trees. The action taken to use genetic identification on the AL in Ontario was a great inspiration to the goal of this project here. One Dr. Retnakaran, has begun to produce molecular markers to which an AL beetle can be identified (Baeta, 2004). Mirroring a criminal investigation, he is working to find the DNA sequence of the AL beetle (Baeta, 2004). Fellow colleagues in the Canadian Forest Service agree that a genetic identification would easily pinpoint the AL beetle and at the same time save many helpless trees (Baeta, 2004). Dr. Retakaran has been successful in his attempts with adult beetles and larva therefore Emerald Ash Borer larva (Figure 1) was used in this experiment. It was an intention to bring about similar results to aid in the ongoing problem of the Emerald Ash Borer invading all of North America 's native ash trees. (Herms, D. A., 2003).

The design of a pesticide is crucial to its affectability. For the EAB researchers, this development is still in its early stages. Systemic pesticide treatments (using products with chemicals such as imidacloprid and bidrin) are being used in Michigan to trees that are no more than 50 % dead. Unfortunately, it is too early yet to say how effective these treatments are. (Anonymous, 2005). This process of finding an adequate insecticide could be greatly added by more studies of the genomic qualities of an EAB.

The initial hypothesis predicted that using the DL7, DL8, DL9, and DL10 primers during PCR would amplify the EAB extracted DNA. Testing of this hypothesis was done with two extractions of DNA, three trials of the PCR and three gel electrophoreses. Unfortunately no helpful results were found. The following paragraphs examine each step closer to find discrepancies and insights into this experiment.

DNA Extraction: A closer look

Extraction of the DNA was done using a replicable protocol as mentioned in Methods, which was done without problem. The first extraction was not successful due to an error in administering RNA-ase to rid the solution of RNA and leave DNA. The second extraction however was successful (Figure 3.). The confirmation was done using the fluorometer using absorbance of light. The extraction produced three tubes of DNA to work with. The fluorometer gave readings to find ratios for each tube. The first tube of, had a ratio of 2.044. Tube two, gave a ratio of 2.83 and tube three had 2.047. To find DNA that is suitable for PCR, it must be between 1.8 and 2.0. The closer a ratio is to 2.0 the greater chance it has adequate DNA to work with. The first tube was the best sample to work with and the third tube was just as close to 2.0. The second tube however was not as close to the ratio that is needed. This could be due to an abundance of RNA in the solution.

PCR process and effect on gel electrophoresis: examined

The PCR procedure was where most of the unsuccessful results can be traced to. The first trial of PCR used primers DL7, DL8 and DL9, respectively in each tube of EAB DNA. Each tube contained 50 µl of solution and the annealing temperature was at a standard 36 °C. It was unsuccessful in the gel electrophoresis however. The gel showed only the ? ladder as producing bands (Figure 4). No other wells that were loaded were evident on the gel. Error is evident in the measurements of each tube in PCR. All of the volume measurements that were called to be mixed in the PCR tubes were below 20µl. This produces a problem due to pipettes that are available in the lab are calibrated to administer 20 µl or higher. When the marker is set to anything lower, the pipette can no longer be considered accurate. During the first gel electrophoresis, the three EAB tubes were run beside a ? ladder at 106 volts for 20 minutes and then at 119 volts for 10 minutes. Another error had occurred here, and especially since it was the first gel run, the loading of the wells was not familiar yet. It can be seen in Figure 4, that the wells were injected with too much solution. The evidence of this is, leaking of the ? ladder wells. Two ladders were used, but a third one can be seen between the two wells, due to leakage. This could have been occurring also in the EAB DNA wells. The absence of DNA bands can be directed to incorrect PCR but at that time it was then questioned as to whether or not DNA was actually present.

Trial two of PCR was done but first the confirmation of DNA was done another way. Ethidium Bromide is known to bind to DNA in solution. It contains a planar group that intercalates between the stacked bases of DNA (Khra et al. 2005). Therefore in the presence of DNA and under ultraviolet light, the solution that have DNA will fluoresce orange. Making a longer procedure short, approximately 2 µl of the supposed EAB DNA solution and diluted ethidium bromide were placed directly onto the UV light in two spots. A positive (potato) and negative (distilled water) control were used next to each. This was done for the three Eppendorf tubes of supposed EAB DNA. All three were positive in DNA presence (Figure 3). Back to PCR at its second trial, DL7, DL8, DL9 and DL10 were used in a 50 µl measurement. The addition of another primer was needed in case the chance that the DL10 would produce results. And a positive control of potato DNA was added. Unfortunately again, no results were evident. Even more disappointing was the absence of bands of DNA from the positive control. This raised suspicions about the solution being used in the PCR process and the annealing temperature at which the PCR machine was set. A temperature set for annealing that is too high, will not allow the primers to bind to the specific sites on the denatured DNA strands.

Trial three can be the point at which major adjustments were made. If the positive control had worked for trial two, then it would have been evident that the primers in use were not applicable to this experiment, but that will be discussed later on. The concentration of DNA was raised, which was done by decreasing the amount of PCR solution to 25 µl. Once again DL7 through DL10 were used individually and combined. Two positive controls were used; E. coli bacteria and P.S. aeruginosa . Both were given primers DL7 and DL8. The abundance of positive controls used was a way to ensure the PCR method was working correctly. Also, the annealing temperature was lowered to 30°C in the PCR machine. It was just another way to define any areas where error had occurred. The end result had the ? ladder and the positive control of P.S. aeruginosa showing bands of DNA (Figure 5). Therefore, the PCR was done correctly, but the mixture of the primers in the E. coli and EAB DNA was not confirmed. To see the primer work with a bacterium such as E. coli, can be surprising. But this can point to similarities between plants and bacteria, as opposed to plants and insects, due to the primers being designed for plants.

Final Conclusions:

The absence of DNA bands to make a genomic fingerprint prohibited the second part of the experiment from being done. Without a replicable DNA fingerprint from the EAB, the cockroach DNA fingerprint could not be compared to find differences in uniqueness.

Ultimately, I believe the main reason for the unsuccessful results was the PCR primers paired with the EAB DNA. The primers had previously been tested on plants and not insects; this could be why the primers did not attach at the binding sites on the EAB genome. Insects and plants are very different organisms, therefore it would be no surprise to compare their genomes and see major differences. The primer sequences of DL7- DL10 were probably not found on the DNA of the EAB, which would inhibit the annealing of the two. If PCR primers that are used on insect DNA could be obtained, then perhaps a successful result could be found and the hypothesis supported. But this experiment can be used as a confirmation that inter-species/ kingdom/organism PCR primers are not possible, at least not in the plant/ insect case.