Genomic Fingerprint Assay for Emerald Ash Borer Larvae fails using primers DL7-10 in AP PCR and Gel Electrophoresis

Eric M Rosenbaum

LBS 145
Section Th1
Dr. Luckie
April 28, 2005

Abstract

            The emerald ash borer (EAB) is responsible for the deaths of millions of Ash trees in North America and eradication of the deadly pest needs support.  The goal of this experiment was to create a genomic fingerprint assay capable of identifying and differentiating the emerald ash borer from other beetles both exotic and native.  The fingerprint would be created using arbitrarily primed polymerase chain reaction (AP PCR) and gel electrophoresis.

            DNA was successfully extracted from emerald ash borer larvae but PCR was not.  After varying concentrations of DNA and primers and lowering annealing temperatures no fingerprint was made.  Most likely this is due to the fact that the four primers used, DL7-10 are unable to bind to and amplify the EAB genome.  The methods and logic used in this experiment can be used as a basis for the development of a genomic fingerprint assay using primers known to work with beetle DNA.

Introduction

            North American forests are being devastated by a foreign beetle.  The emerald ash borer (EAB), Agrilus planipennis, found its way to Detroit, Michigan even though it is native to eastern Russia, northeastern China, Japan, Korea, Mongolia, and Taiwan, ã(Herms et al, 2003).ä  The EAB was first found in southwest Michigan in June 2002 and has most likely been living in Michigan for nearly five years prior to discovery, ã(Flint, 2003).ä  Due to the fact that the emerald ash borer is not native to North America it has a highly destructive potential simply because North America has evolved without the EAB leaving it nearly free of natural predators, parasites, diseases, or any defense from the Ash tree, ã(Howse et al, 2002).ä  Other tree borers are less devastating.  Some target mostly sick or very old trees such as Podosesia syringe another ash borer also known as the lilac borer, ã(Halcomb and Hale, 2000).ä 

The EAB lays its eggs in the bark of Ash trees, of the genus Fraxinus, ã(Herms et al, 2003).ä  The eggs develop into larvae that eat away at the vascular cambium tissue under the bark as they tunnel through the trunk, ã(Howse et al, 2002).ä  This severely disrupts the treesâ ability to transport nutrients from the leaves to the roots as well as water from the roots upward, causing the tree to die from the top down, ã(Shetlar, 2001).ä  An Ash tree infested with the EAB will likely die within 2-4 years, ã(Herms et al, 2003).ä  Wood from ash trees is hard and strong which is why it is used for baskets, flooring, furniture, sports equipment, tool handles and more, ã(Howse et al, 2002).ä  Only a year after its discovery it was estimated that the emerald ash borer had caused at least $11.6 million in the state of Michigan alone, ã(Flint, 2003).ä  The EAB is responsible for killing 15 million trees in Michigan and has yet to be stopped, ã(Hawthorne, 2005).ä

Since the discovery of its presence in North America in 2002 much effort has put towards the preservation of North American Ash trees, ã(Baeta, 2004).ä  A thorough understanding of the emerald ash borer is crucial to the defense of the Ash tree.  This experiment was begun to create an assay that can be used to make a genomic fingerprint of the emerald ash borer through arbitrarily primed (AP) polymerase chain reaction (PCR) and gel electrophoresis.  PCR is used to amplify DNA or a specific section of DNA.  Traditionally PCR is performed using primers designed to amplify the desired section of DNA.  However, AP PCR uses arbitrary primers to amplify arbitrary portions of a genome.  In this lab AP PCR was performed using primers DL7, DL8, DL9, and DL10.  Work was done with the hypothesis that at least one of these primers could be used to amplify several portions of the emerald ash borer genome so that electrophoresis could be used to create a genetic fingerprint.

            DNA was extracted from emerald ash borer larvae and quantified using a fluorometer.  PCR was then performed several times under differing conditions with each primer as well as combination of all primers.  Variables included concentration of EAB DNA, primers, and free nucleotides and two annealing temperatures were also used in PCR.  Unfortunately AP PCR on emerald ash borer DNA was unsuccessful using primers DL7-10.  Positive controls were also used of DNA samples known to amplify with the primers used and PCR only worked with a lowered annealing temperature and a higher concentration of free nucleotides.  If AP PCR had worked on the EAB, gel electrophoresis would produce a genomic fingerprint.  Had a genomic fingerprint been made and replicated the same procedure would have been used on another beetle, perhaps a cockroach.  This would be used to confirm that the EAB fingerprint is indeed unique.  A genomic fingerprint of a cockroach should be similar to the emerald ash borer for both are beetles; however they should still be different so that the EAB can be identified by its genomic fingerprint.

Methods

            First, emerald ash borer DNA had to be obtained. Four EAB larvae were received preserved in phenol from the laboratory of Dr. David R. Smitley, professor of entomology at Michigan State University.  These larvae were originally collected in Troy, Michigan in October 2004.  To extract DNA from these larvae a protocol from Alicia Bray, LBS 144 graduate TA was followed.  Alicia Bray also provided solutions X and Y that are required in the protocol.  Solution X was composed of 0.25mL 2M Tris-HCl (pH 8.0), 2.5mL 10% SDS (pH 7.2), 0.6mL 5M NaCl, 5mL 50% sucrose, 1mL 0.5mL EDTA (pH 8.0), and 40.6mL dH₂O.  Solution Y was composed of 7.5mL 2M Tris-HCl (pH 8.0), 3.7mL 10% SDS (pH 7.2), 5mL 50% sucrose, 10mL .5EDTA (pH 8.0), and 23.8mL dH₂O.  In an Eppendorf (EP) tube, 10μL of stock proteinase-K (10mg/mL) was added to 1mL of solution X and vortexed.  In a new EP tube 250μL of the previous solution was added.  A larva was cut in half by sterilized tweezers and half was placed in the EP tube and crushed into a few pieces with the same tweezers.  The EP tube was then incubated at 55¼C for 5 min.  The larva was then ground with sterile tweezers to disintegrate its structure and incubated again at 55¼C for 30 min.  250μL of solution Y was added to the tube before it was incubated on ice for 10 min.  500μL of chloroform/isoamyl alcohol (24:1) was added and the tube mixed, then placed on ice for 3 min.  The tube was centrifuged in a microcentrifuge at approximately 23¼C at 14,000 rpm for 5 min.  The upper aqueous layer was removed and placed in a new EP tube, and 500μL of chloroform/isoamyl alcohol (24:1), mixed, and placed on ice for another 3 minutes.  Te tube was centrifuged at about 23¼C at 14,000 rpm for 5min and the upper aqueous layer added to a new EP tube.  500μL of chloroform isoamyl alcohol (24:1) was added, the tube vortexed, and centrifuged again at 23¼C at 14,000 rpm for 5 min.  The upper aqueous layer was removed and placed into a new EP tube and 50μL of 5M ammonium acetate and 1mL of chilled ethanol (95%) were added.  5M ammonium acetate was prepared by adding 19.25g of solid ammonium acetate to 30mL dH₂O and then the volume was adjusted to 50mL.  The tube was then inverted 10 times and placed in a freezer overnight.  It was then centrifuged at approximately 23¼C at 14,000 rpm for 25 min.  The supernatant was discarded and 50μL of 95% ethanol added to the pellet and centrifuged at 14,000 rpm for 5 min.  Excess ethanol was removed and the tube was set open, upside-down allowing the pellet to dry.  The pellet was then resuspended in 24μL of 1x TE.  This extraction protocol was performed four times using a total of two larvae.

            The next step was to determine how much DNA had been extracted as well as its purity.  30μL of a DNA sample was added to 1.47mL TE and vortexed.  The sample was then tested with a fluorometer at wavelengths of 260nm, 280nm, and 310nm.  From this data, purity and concentration of DNA can be calculated.  After it was confirmed that a good concentration and purity of DNA had been obtained, PCR was performed.

            PCR was performed first using three primers individually, DL7 - CAATCGCCGT, DL8 - CAGCACCCAC, and DL9 - TCACCACGGT (all presented 5â to 3â).  DL10 which was later used is GGATATGCCG.  First 22μL of EAB DNA was added to an EP tube.  Then 12μL free nucleotides, 15μL of 10x NH₄ reaction buffer, 7.5μL MgCl₂, 1μL of Taq Polymerase, and 86.5μL of water were added to the EP tube.  This ãPCR cocktailä was then divided among 3 PCR tubes, each containing 48μL.  To each PCR tube containing 48μL PCR cocktail, 2μL of one primer was added.  DL7 was added to one EP tube, DL8 to another, and DL9 to the last.  The tubes were then placed into the PCR machine which was set for 45 cycles at 94¼C to denature, 36¼C for annealing, and 72¼C for extension.  Once the PCR machine was finished it cooled the samples to 4¼C and then the tubes were removed and frozen overnight.

            Gel electrophoresis was performed using methods described in Mini-Maniatis, ã(Fritsch et al, pp6.9-6.16)ä using 2μL of loading dye and 18μ of PCR mix and Lambda Ladder was used for reference.  The gel only produced bands from the ladder so the DNA from extraction was tested to make sure that the fluorometer responded to DNA and not possible impurities.  To do so 10μL ethidium bromide was added in 4 spots to the ultraviolet light.  One was used as a negative control to which 10μL water was added and another was used as a positive control to which potato DNA was added.  The other two were each used to test the DNA from one tube of extracted DNA; 10μL was added to each remaining spot of ethidium bromide.  This was done three times, once for each tube/trial of DNA extraction.

            PCR was performed again, this time the PCR cocktail was prepared for 4 trials of EAB DNA and 1 primer (1 trial per primer) and two more PCR tubes were used, one for all primers and another for potato DNA and DL7.  Ultimately 5 PCR tubes each had 8μL EAB DNA, and one had potato DNA.  Also each tube had 8μL free nucleotides, 5μL NH₄ reaction buffer, 5μL MgCl₂, and .2μL Taq polymerase.  2μL per primer per tube was added and then water to total 50μL per tube.  Then PCR was run but this time at a lower annealing temperature of 30¼C.  Gel electrophoresis was performed again, this time using 1Kb Ladder.

            PCR was performed again this time per tube using only 4μL of free nucleotides, 2.5μL NH₄ reaction buffer, 2.5μL MgCl₂, and each tube totaled 25μL.  5μL of DNA was used per tube and no change in amount of other components.  Eight trials were performed, one of E. coli with DL7 as a positive control and another with DL8, another positive control of Ps aeruginosa and DL7, one trial of EAB with all four primers, and four more EAB trials each with one of DL7-10.  Again the annealing temperature was set to 30¼C.  Gel electrophoresis was performed again, this time splitting the trials on to two gels and using 1Kb ladder on both.

Results

            Extraction of DNA from the emerald ash borer larvae worked successfully.  Using the fluorometer it was determined that it was in good concentration as well as purity, ã(Table 1).ä  The purity values are a ratio.  A ratio is between 1.8 and 2 is ideal and suggests that there is DNA of a good purity for PCR.  If the value is higher than 2 than the DNA sample is likely contaminated with RNA.  Extraction trial 2 produced a high purity ratio.  However trials 1 and 3 produced ratios very close to 2 (2.045 and 2.053) and are considered to be a purity that can be used for PCR.

The first trial of PCR was unsuccessful.  This was made known after performing gel electrophoresis; only the ladder appeared, ã(Figure 1).ä  Next each tube was tested for the presence of DNA by using ethidium bromide and an ultraviolet light.  It showed that each tube did contain DNA, ã(Figure 2).ä  The second trial of PCR was no more successful than the first and could be seen from gel electrophoresis, ã(Figure 3).ä  The third trial of PCR showed that PCR was being performed properly.  This could be seen by the gel electrophoresis for Ps aeruginosa, one of the positive controls did amplify and the ladders can also be seen.  However the E. coli positive controls did not amplify, nor did the emerald ash borer DNA, ã(Figure 4).ä

Discussion

            The findings of this experiment negate the hypothesis that a replicable genomic fingerprint assay can be developed for the emerald ash borer using primers DL7-10 in AP PCR and gel electrophoresis.  The data suggests that different primers are required and even perhaps that traditional PCR would be more affective than PCR using arbitrary primers.

            After DNA was extracted using the protocol obtained from Alicia Bray, a fluorometer was used to quantify and test the purity of each DNA sample.  Calculations from the fluorometer indicate that one of the DNA samples was slightly contaminated with RNA.  Most likely this was the result of the stage of the extraction protocol in which the DNA pellet is rinsed in chloroform/isoamyl alcohol several times.  Perhaps the pellet was not mixed enough in the alcohol solution.  If necessary this sample would have been rinsed with alcohol several times and then tested with the fluorometer again.  However, the other two samples were ready to be used.  The next step was PCR.

After completing PCR using DL7-9 it gel electrophoresis was performed and it became apparent that something had not worked at any given stage of the experiment.  Even though the fluorometer readings and calculations suggested that a good sample of DNA had been extracted it was possible that those readings were false.  The fluorometer simply measures how much light passes through a sample and it could be possible to get similar readings due to substances other than DNA.  Due to this reasoning another test had to be performed to determine that the DNA extraction protocol had in fact extracted DNA.

            To test for the presence of DNA the ultraviolet light was used.  Ethidium bromide sticks to DNA between base pairs and fluoresces in the presence of ultraviolet light, ã(Fritsch et al, pp6.6).ä  By taking advantage of the chemical properties of ethidium bromide it was determined that there was indeed DNA extracted from the larvae.  The next place to look for error was the polymerase chain reaction.  At this stage it seemed very possible that either human error during the preparation for PCR had occurred or the primers used do not match the emerald ash borer genome in such a way as to amplify portions of the genome.

It became evident at this stage that the first trial of PCR should have been performed with a positive control for at least one of the primers used.  So to rule out human error PCR was performed again under similar circumstances with potato DNA as a positive control for it is known to amplify in PCR with any of the available primers.  However, under the suspicion that the primers may not amplify the EAB DNA, a lower annealing temperature was used to increase the chances that the primers would bind to the EAB genome.  Also DL10 was used as well as a trial with all four primers.  This was done incase primers DL7-9 would not amplify the EAB DNA.  It was hoped that DL10 would work even if the other primers did not.  Using all four primers at once was done because it is possible that two of the available primers would amplify the EAB DNA.  Perhaps two primers were able to bind to the DNA but were individually unable to amplify the DNA but together amplification could be possible.  If the trial of all four primers had amplified, combinations of only two primers would have been run through PCR to find the two primers that were working.  It is even possible that amplification could have only worked with all four or even three of the primers.  This would have been discovered by the testing of several combinations of primers.  Instead none of the trials including the control amplified.  This suggested that human error was at play.

PCR was performed a third time with more alterations to the procedure.  Less of everything except DNA and primers were used to increase the concentration of both without depleting the DNA source.  Perhaps the primers could work if there was more to work with.  However this thought process is flawed.  A higher concentration of free nucleotides should have been used as well.  Nonetheless the concentration of free nucleotides did not decrease and therefore should have been sufficient to support amplification.

Amplification of EAB DNA did not occur, nor did the E. coli positive controls. However Ps aeruginosa did amplify which indicates that PCR was performed properly.  Even though there was some minor success with PCR there is still something amiss.  First of all a positive control failed with two different primers.  Further testing could be done by running PCR again with E. coli and Ps aeruginosa.  If the same results occur than that would suggest that one of the samples is contaminated.  Either the E. coli DNA is contaminated with something to prevent amplification of its DNA or the Ps aeruginosa DNA is contaminated with something that would be seen through electrophoresis even without PCR amplification.  The later could be tested by running the Ps aeruginosa DNA in gel electrophoresis without running PCR.  If a band appeared than the DNA is contaminated.  This is definitely a possibility due to the fact that only one band appeared.  However it is possible that the primer DL7 amplified one large region of DNA that was too large to move far through the agarose gel.

This assay failed but could be completed successfully with primers designed for beetles.  This would allow for the fingerprinting of many beetles for comparison to the emerald ash borer.  This would be especially useful in identifying exotic beetles that begin to spread in North America or even other tree borers native to North America.
 

References

Baeta, D. 2004. Tracking a tiny but deadly menace. ÐÐCatalyst. http://www.carleton.ca/catalyst/sf/db/dkb-beetle.html Accessed 3/22/05.

Flint, T. 2003, May. Emerald Ash Borer, Michigan Eradication Strategy.

http://www.michigan.gov/documents/MDA_EAB_Strategy_86341_7.pdf Accessed 4/18/05.

Fritsch, Maniatis, and Sambrook. Molecular Clonig 2^nd Edition. 1:6.9-6.16

Haack, R. A. et al. 2002. The emerald ash borer: a new exotic pest in North America. Michigan Entomological Society 47.3 1-5

Halcomb, M. A., Hale, F. A. 2000. Common Tree Borers in Tennessee. Agricultural Extension Service. The University of Tennessee. http://www.utextension.utk.edu/publications/spfiles/sp547.pdf Accessed 4/22/05.

Hawthorne, M. 2005, April 19. Have we finally squashed the beetle? Chicago Tribune http://www.chicagotribune.com/news/local/chi-0504190132apr19,1,4804550.story?coll=chi-newslocal-hed Accessed 4/26/05.

Herms, D. A., Stone, A. K., Chatfield, J.A. 2003. Emerald Ash Borer: The Beginning of the End of Ash in North America? Ornamental Plants Annual Reports and Research Reviews. Ohio Agricultural Research & Development Center.

Howse, G. M, McCullough, D. G., Scarr, A. T. 2002, July. Emerald Ash Borer. Forest Health Alert 3 Canadian Forest Service.

Shetlar, D. G. 2001, Oct. Ornamental Pests: Borers. Entomology 462. Ohio State University. http://bugs.osu.edu/~bugdoc/Shetlar/462/pdf/Ent462Oramentalborersbw.PDF Accessed 4/25/05.

Figures

Figure 1.  Gel electrophoresis trial 1.  Wells 1 and 2 are Lambda Ladders and the vague bands in between them are the result of well 1 spilling over.  3, 4, and 5 are EAB DNA primed with DL7, DL8, and DL9 respectively.  The lack of fluorescent banding by wells 3-5 suggests that PCR was unsuccessful at amplifying the EAB genome.

Figure 2.  Test for the presence of DNA after extraction.  On an ultraviolet light, 10μL of ethidium bromide was placed in four spots (1-4).  10μL of distilled water was placed on location 1 and 10μL of potato DNA was placed on location 2 as a control.  On locations 3 and 4 10μL emerald ash borer DNA was added as two trials.  The EAB DNA came from tubes 1-3 which correspond with A-C.  Pink signifies the presence of DNA.

Figure 3.  Gel electrophoresis trial 2.  Well 3 is the 1Kb ladder and the only DNA that banded.  Well 1 is potato DNA used as a positive control.  The fact that this did not band suggests that PCR was conducted improperly.  Well 2 contained EAB DNA primed with all four primers, DL7-10.  Wells 4-7 contained EAB DNA and one primer each, DL7-10 respectively.

Figure 4.  Gel electrophoresis trial 4.  Well 4, positive control Ps aeruginosa shows banding.  This indicates that PCR was performed properly.  However, wells 1 and 2 are E. coli DNA and also positive controls but tested negatively.  This may be the result of many possibilities (see discussion for details).  Wells 3 and 8 are 1Kb ladders.  Well 5 is EAB DNA primed with all four primers.  Wells 6, 7, 9, and 10 are EAB DNA primed with DL7-10 respectively.

Tables
 

Table 1.  EAB DNA fluorometer readings and calculations.  Purity is calculated from the following equation:  Concentration is calculated from: