RAPD PCR using Primers DL7, DL8, DL9, DL10 and Gel Electrophoresis with Emerald Ash Borer for Genomic Fingerprinting

Jillian K. Trombley

LBS 145

Section Th1

Dr. Luckie

April 27, 2005

Abstract

In order to preserve the Ash trees that are found in most of North America and Canada , information must be gathered about the Emerald Ash Borer (EAB). A defense against the invasive species has been launched by identifying the insect on a genetic level. The purpose of this experiment is to extract DNA from the EAB, amplify it, and produce a unique replicable gel using gel electrophoresis for identification. Mature Ash Borer larva stored in alcohol was used to extract DNA. A protocol specifically for Alcohol-Preserved insects was used. Once confirmed that DNA was successfully extracted, three trials of polymerase chain reactions (PCR) were used to cut and amplify the DNA. The first trial used three vials of Emerald Ash Borer DNA with primers DL7, DL8, and DL9. No positive control was used. The EAB DNA did not produce any bands of DNA during gel electrophoresis. The second trial added the EAB DNA to the primers DL7, DL8, DL9, and DL10. One vial held EAB DNA with all four primers. The positive control, Potato DNA, was added to primer DL7. During gel electrophoresis, no bands of DNA were produced. During the third trial of PCR, three positive controls were used; E. coli with primer DL7, E. coli with primer DL8, and P.S. aeruginosa with primer DL7. The EAB DNA was added to Primers DL7 through DL10. One vial held EAB DNA with all four primers. Only the P.S. aeruginosa DNA produced bands of DNA during gel electrophoresis.

Legend

Figure 2. Gel Electrophoresis producing DNA bands from Eppendorf tube 1. Emerald Ash Borer DNA was extracted into three Ep tubes, DNA from the first is shown along with a negative control (water) and a positive control (Potato DNA). The ratio of DNA given by the Fluorometer was 2.044. The ratio was close to 2, which signifies that it is Emerald Ash Borer DNA in the tube. The DNA was then mixed with ethidium bromide and placed on the UV light. If bands showed under the UV light than ethidium bromide was clinging to DNA (Numbers 2, 3, 4) and extraction was successful. The amount of DNA present in this Ep tube was 1.1204 µg/mL.

Discussion

It was predicted that the primers DL7, DL8, DL9, and DL10 combined with Emerald Ash Borer DNA used in PCR would produce bands of DNA when run through gel electrophoresis. This was predicted because PCR has been used successfully in the past with invasive beetles to produce a unique genomic fingerprint. A similar infestation occurred in Ontario , Canada with the Asian Long Horned Beetle. The Asian Long Horned Beetle bores into Maple trees and lays larvae (Baeta, 2004). During the infestation little was known about the Asian Long Horned Beetle (Baeta, 2004). This prompted scientists to take a DNA fingerprint of the beetle so that it could assist in identifying this beetle from other beetles in further infestations (Baeta, 2004). If DNA from the Emerald Ash Borer larvae (Figure 1) could be identified through gel electrophoresis, it would provide scientists with enough information to identify the beetle if it continued to spread.

Since ash trees are the most commonly planted trees around homes and urban forests, it is likely the Borer will continue to invade (Herms, D. A., 2003). The cost of waging a war against these insects has already taxed Michigan and the surrounding states economies. This is not including the cost of having to quarantine the ash wood, and no longer being able to use it. (Herms, D.A., 2003). The Ash Borer is posing serious threats to both the environment and the economy. Proper identification of the insect will provide more accurate treatment of eradication, as the most effective pesticides can be used (Herms, D.A., 2003).

Emerald Ash Borer DNA Extraction

The protocol used for extraction was followed closely with few alterations made. During extraction, DNA was collected into three Ep tubes. It was determined that DNA was present in the tubes by using a fluorometer and gel electrophoresis. The fluorometer quantized the DNA and produced a ratio number. If it was between 1.8 and 2, then DNA was present. If the ratio number was greater than 2 then the DNA present was probably due to RNA. The first Ep tube of EAB DNA had a ratio of 2.044 on the fluorometer. Since it was so close to 2, the DNA is believed to be DNA from the Emerald Ash Borer. The second Ep tube of EAB DNA had a ratio of 2.83. This was slightly more than 2 therefore the DNA present could have actually been RNA. The third Ep tube of EAB DNA had a ratio of 2.047, which was also close enough to 2 to be considered actual DNA present. Another way to ensure that DNA was present was to place diluted ethidium bromide directly on the UV light in three separate places. The three tubes of Emerald Ash Borer DNA were then added individually to the ethidium bromide. The ethidium bromide clings to the DNA and glows when the UV light is turned on. The first Ep tube showed glowing spots of DNA (Figure 2). The second Ep tube also showed bands of DNA (Figure 3), as did the third Ep tube (Figure 4).

PCR Analysis of Trials One, Two, and Three and Gel Electrophoresis

Trial one of the polymerase chain reaction produced no DNA amplification. Since the trial did not include a positive control, no conclusive fingerprint could have been produced. A positive control shows that PCR was run correctly and the DNA bands are not a mistake. In trial one the primers DL7, DL8, and DL9 were each placed in a vial with the PCR solution and put in the PCR machine. Each vial held a total of 50µL including primers and DNA. RAPD PCR was used so that each primer would amplify different, arbitrary parts of the DNA. The annealing temperature was kept at 36° C; this affects how often the primer binds to the DNA. It was not lowered until trial three. The three vials of DNA were then run through gel electrophoresis, along with a Lambda/hind III Ladder (Figure 5). The purpose of the Lambda/hind III Ladder was to serve as a comparison between unknown base pair sizes of Emerald Ash Borer DNA to known base pair size. The shorter chains of DNA will travel further than the longer chains because they can easily move around large chunks of agarose in the gel. In this gel the Lambda/hind III Ladder was placed into two separate wells. Some of the ladder leaked into the well between the two, producing the faint ladder between them (Figure 5). The gel electrophoresis was run at 106 volts for approximately 20 minutes and then at 119 volts for approximately 10 minutes The Emerald Ash Borer DNA with primer 7 did not produce any bands of DNA, nor were any bands produced with primers 8 and 9 (Figure 5). Without the use of a positive control in this trial it is impossible to determine the cause of the missing DNA bands. The DNA bands may have been absent because the primers did not anneal or an error could have occurred when the PCR solutions were being made.

During trial two of PCR, the methods were improved and more measures were taken to ensure that the PCR solutions were made properly. A positive control, Potato DNA was added to the vials undergoing PCR. In this trial, 6 vials were placed in the PCR machine. Each vial held a total of 50µL including primers and DNA. The first four vials each held Emerald Ash Borer DNA and one primer: DL7, DL8, DL9, or DL10. A fifth vial held EAB DNA with all of the primers mixed together and a sixth vial held the positive control, Potato DNA, with primer 7. The potato DNA was known to produce bands of DNA with primer DL7. Along with a Lambda/hind III ladder, the vials were then run through gel electrophoresis at 106 volts for approximately 20 minutes and then at 119 volts for approximately 10 minutes. Only the Lambda/hind III ladder produced DNA bands (Figure 6). The positive control failed to produce DNA bands and the Emerald Ash Borer DNA with individual primers and all of the primers produced no DNA bands (Figure 6). This could have occurred because of an error or miscalculation when determining the amounts of chemicals to use in the PCR solution. Too much or too little of a chemical, specifically DNA could have affected the primers annealing. If there is too little DNA, the primers will not bind. The temperature also could have prevented the primers from binding. Too high of temperature will prevent the primers from annealing. Since the positive control did not produce any DNA bands, PCR was most likely done incorrectly. The specific primers being used could have also played a role in the lack of a genomic fingerprint. The primers DL7 through DL10 were all created purposely for the LBS 145 lab; however the lab is geared towards the extraction and amplification of plant DNA. Since the specimen used in this experiment is an insect, there was never a guarantee that the primers would bind to the DNA.

In trial three more alterations were made to the PCR protocol. The amount of PCR solution in each vial was reduced to a total of 25 µL including primers and DNA. This was done to increase the concentration of DNA and the chances of the primers annealing to occur. The primers DL7, DL8, DL9, and DL10 were all used individually with the Emerald Ash Borer DNA. A fifth vial contained EAB DNA with all four of the primers. A sixth vial contained E. coli DNA with the primer DL7. A seventh vial contained E. coli DNA with the primer DL8 and an eighth vial contained P.S. aeruginosa with primer DL7. Vials 6, 7, and 8 all served as positive controls. The number of positive controls was increased for this trial to ensure that the PCR protocol was being performed correctly. Also some of the positive controls were not working correctly with the primers. DNA bands were not being produced when the procedures were carried out correctly. In this trial the annealing temperature of the PCR machine was also reduced to 30° C. Reducing the annealing temperature increases the chances that the primers will bind to the DNA (Trombley, 4/18/2005) The vials were then run through gel electrophoresis on two separate gels. The first gel contained the three positive controls and EAB DNA with all of the primers DL7-DL10 added. The Lambda/hind III ladder produced bands of DNA and the positive control P.S aeruginosa with primer DL7 produced faint bands of DNA (Figure 7). The E. coli DNA with primer 7 and with primer 8 produced no DNA bands. The EAB DNA with all of the primers together produced no DNA bands. The E. coli may not have produced bands of DNA because of an error in the PCR solution. The amount of the primer DL7 used may have been too little or the primers may have been unable to find the correct binding cites . The second gel held the EAB DNA with the individual primers DL7-DL10 added. Only the Lambda/HIND III ladder produced bands of DNA. None of the EAB DNA with the individual primers produced bands of DNA (Figure 8). Since the positive control, P.S. aeruginosa with primer DL7, produced DNA bands, the PCR protocol was successful (Figure 7). The lack of DNA bands being produced by the Emerald Ash Borer larvae are most likely caused by a variety of mishaps. It would be difficult to narrow the failure to simply one reason. The temperature of the PCR machine, concentration of DNA or primers, or the primers themselves could have been the cause. However had primers specific to the EAB chromosome been synthesized, it is predicted a genomic fingerprint could have been produced.