Determining canine breed based on DNA bands Katie Vickers 2006-2007 Senior Capstone

1. Determining canine breed based on DNA bandsKatie Vickers2006-2007 Senior Capstone Results The first gel produced contained 3 of the PCR reactions with a different primer used for each reaction, but there were no visible DNA bands. For the second gel’s samples, the DNA added to PCR was increased from 1 μl to 2 μl to ensure enough DNA was added to the reaction, however there were no visible bands. For the third gel’s samples, only primer 1 was used and the annealing temperature was changed from 60°C to 58°C ( Francisco et al., 1996). Nine out of the 10 samples loaded on the gel had visible DNA bands with all of the bands approximately the same distance from the wells (See Table 1). The same conditions were used for gel 4’s samples, except only primer 2 was used. This gel had 6 out of the 10 samples with visible DNA bands and again all the bands were about the same distance from the wells (See Table 2). Finally, only primer 3 was used for the fifth gel, however no DNA bands were seen. A multiplex (using many primers in PCR) was tried using all of the primers for the 6th gel because of Koskinen and Bredbacka’s (1999) experiment, and no visible DNA bands were seen. Gels 7 and 8 had a mixture of all different samples because the PCR machine is able to hold more samples than can be tested on one electrophoresis gel. These two gels contained the following samples: primer 1 used on 4 fresh samples (collected since not getting results at the time), 2 samples from the first PCR run made, 3 samples from when primer 2 was used, 3 samples from when primer 3 was used the first time, and 7 samples from when primer 3 was tried for the second time. Out of all these samples tested, 2 of the fresh samples and 2 of the primer 2 samples had visible DNA bands (See Table 3). Introduction Although not much research has been performed using canine DNA, there has been increasing motivation for canine breed identification. One reason for this increasing motivation to test canine DNA is to preserve the genetic integrity of rare breeds against inter-breeding (Randi & Lucchini, 2002). Another reason is to identify certain breeds such as Pit Bulls that are banned in some areas (Blackman, 1999). Instead of just looking at a dog and suspecting it might be a Pit bull, DNA tests could determine that a dog is indeed a Pit Bull. Detecting the unsanctioned use of breeds that are faster in pure breed races is an additional motive for breed identification because an owner could have an unfair advantage by entering a greyhound in a race that was specifically for whippets (Coppinger, 1991). However one of the most important uses of canine DNA is in forensic crime labs (Aaspollu & Kelve, 2003; Schneider et al.,1999). A case in Estonia used canine DNA in order to determine which dog (s) attacked and killed a woman (Aaspollu & Kelve, 2003). Reference samples from neighborhood dogs were compared to the samples found at the scene, however there no match was found (Aaspollu & Kelve, 2003). In another case, a dog that was suspected of causing a traffic accident had his DNA collected and compared to samples gathered from the scene (Schneider et al., 1999). The dog was excluded as a suspect because his DNA did not match that found at the scene (Schneider et al., 1999). Since DNA would be a vital tool for breed identification, it needs to be experimented on so that more can be learned about it, which is why this is project was conducted. The hypothesis is that canine DNA bands would be different distances from the gel wells in order to determine canine breed. Soft-Coated Wheaten Terrier Mutt Soft-Coated Wheaten Terrier American Cocker Spaniel Soft-Coated Wheaten Terrier American Cocker Spaniel Golden Retriever Golden Retriever Brittany Spaniel Gel 4: Gel of PCR reactions of various dog breeds. Annealing temperature of 58°C was used. See procedures for other PCR conditions used. Future Experiments There are a variety of future experiments that could be attempted in order for canine breed to be verified by DNA. Different primers than the ones used in this capstone project could be used in order to see if canine breed can be identified based on DNA. Francisco et al. (1996) along with Koskinen and Bredbacka (1999) tested many more primers than I was able to test, so selecting different primers or using more primers might allow canine breed to be identified. Selecting different breeds than what were tested in this capstone could also be an option. Since MetaMorphix and Mars Veterinary came out with their breed identification tests too late for them to be of use for this capstone, perhaps getting in contact with one or both of these companies could provide some insight into the procedures and materials that they used in order for breed identification to be possible for someone else to try. Although the references used in order to obtain procedures dealing specifically with canine DNA did not mention using restriction enzymes, perhaps they should be used. Every individual has their own unique number of repeats where a restriction enzyme would cut, perhaps there are repeat sequences in dogs that might provide the ability to differentiate breed. Procedures Saliva samples were collected from 12 Golden Retrievers, 12 Labrador Retrievers, 9 American Cocker Spaniels, 5 Brittany Spaniels, 2 Soft-Coated Wheaten Terriers, and 1 mutt for a total of 41 individuals using sterile cotton swabs (Brown, 2006). The DNA was extracted from the swabs using TN buffer and was purified using phenol/chloroform extractions (Brown, 2006). The DNA was then prepared for PCR using the Taq buffers, nucleotides, primers, and Taq polymerase (Brown, 2006). The primers were named 2001, 2132, and 2088 in the reference they were found in and were renamed primers 1, 2, and 3 respectively for this experiment (Francisco et al., 1996). PCR was run for 35 cycles with the following conditions: Initial denaturation for 3 min. at 94°C, denaturation for 30 sec. at 94°C, annealing for 1 min. at either 58°C or 60°C, extension for 1 min. at 72°C, and final extension for 15 min. at 75°C with the machine cooling to 4°C (Koskinen & Bredbacka, 1999; Francisco et al., 1996). There were two different annealing temperatures used in order to optimize for the different primers that were used in this experiment (Koskinen & Bredbacka, 1999; Francisco et al., 1996). The DNA was loaded into the agarose gel wells, and electrophoresis was performed for 2.5-3 hours on a voltage that ranged from 59-63 volts (Brown, 2006). The gels were then examined for bands on the UV transluminator, and the distance that the DNA bands traveled from the well was measured using a ruler and recorded. Table 3: Distance of the DNA bands from the gel wells on gels 7 and 8 Table 1: Distance of the DNA bands from the gel wells on gel 3 with primer 1 used Table 2: Distance of the DNA bands from the gel wells on gel 4 with primer 2 used Acknowledgments Dr. Tschunko, capstone advisor Dr. Brown, “unofficial capstone advisor” Dr. Hogan, capstone class professor and academic advisor All the dogs that cooperated when I collected samples Discussion/Conclusions Primer 3 was tested on 17 samples, and did not produce any DNA bands. The annealing temperature used by Francisco et al. (1996) was 58°C, but perhaps the annealing temperature needed to be lowered because the primer was not able to bind to complementary sections of the DNA. Primers 1 and 2 worked when the annealing temperature was 58°C, but the DNA bands were approximately the same distance from the wells for all the samples. Different primers might be the key to being able to identify canine breed based on DNA bands. Since the DNA bands were the same distance from the gel wells for all of the breeds tested, it was not possible to determine canine breed based on DNA bands using these primers. Although in this experiment it was not possible to determine canine breed based on DNA bands since the DNA bands were the same distance from the wells for all of the breeds, new DNA testing in order to determine canine breed has been developed by MetaMorphix and Mars Veterinary (Mott, 2007). MetaMorphix introduced their Canine Heritage test in February 2007 making it the first DNA-based diagnostic test to identify dog breeds (Mott, 2007). All that they require is a saliva sample be sent and they are able to identify 38 breeds (Mott, 2007). Mars Veterinary will launch its Wisdom Panel MX mixed breed analysis test in the summer of 2007 (Mott, 2007). They claim to be able to identify over 100 breeds (Mott, 2007). References Aaspollu A, Kelve M. 2003. The first criminal case in Estonia with dog’s DNA data admitted as evidence. International Congress Series 1239: 847-851. Blackman D. 1999. Practicality of breed specific legislation in reducing or eliminating dog attacks on humans and dogs. Article available at http://www.dog-play.com/pitbull.html. Brown D. 2006 Molecular Biology Lab Manual. Marietta, OH. Marietta College. Coppinger R. 1991. Can you I.D. your dog with DNA? Available on-line at: http://www.grapevine.net/~wolf2dog /dnaid.html. Francisco LV, Langston AA, Mellersh CS, Neal CL, Ostrander EA. 1996. A class of polymorphic tetranucleotide repeats for canine genetic mapping. Mammalian Genome 7: 359-362. Koskinen MT, Bredbacka P. 1999. A convenient and efficient microsatellite-based assay for resolving parentage in dogs. Animal Genetics 30: 148-149. Mott, Maryann. 2007. Want’s in the mix? Find out with a DNA test. Dog Fancy, May, 10. Randi E, Lucchini V. 2002. Detecting rare introgression of domestic dog genes into wild wolf (Canis lupus) populations by Bayesian admixture analyses of microsatellite variation. Conservation Genetics 3:29-43. Schneider PM, Seo Y, Rittner C. 1999. Forensic mtDNA hair analysis excludes dog from having caused a traffic accident. International Journal of Legal Medicine 112: 315-316. PCR machine Electrophoresis unit