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Microsatellites as Identifiers

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This is a presentation made for the laboratory component of my Spring 2015 Genetics course. In this presentation, we identify individuals' microsatellite sequences and use that information to determine paternity. Interestingly, though the three of us are definitely not related, our microsatellites showed that my two lab partners could have been my parents, supporting the idea that more loci need to be examined to truly determine paternity/maternity.

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Microsatellites as Identifiers

  1. 1. Maternity and Paternity Testing Using Microsatellites for Identification Julia MacDougall, Andrew Long, and Sarah Lerwick
  2. 2. The nature of microsatellite variation ● STRs of 2-5 base pairs ● High variability from “slippage”
  3. 3. Microsatellites as tools for analysis ● Highly variable nature ● Gene duplication & deletion ● Marker assisted selection ● forensics ● *Paternity and maternity testing
  4. 4. Accuracy of Microsatellite Analysis: Disputed Paternity in Humans ● Ain Shams Univ. and Dept. of Forensic Evidence in Cairo, Egypt ● Swiss & Spanish collaboration ○ Mutation analysis ○ Able to explain variations from expected
  5. 5. Microsatellite Application: Paternity Testing for Ecological Conservation ● Korean native horse ● Endangered ● Important for conservation
  6. 6. Microsatellite Markers and Primers
  7. 7. Case Study Question ● It is known that S3-3 is the mother of S3-5, but there is question of who the father is. ● The mother thinks the father is either S3-4 or S3-6. ● Can we tell conclusively from this data which person is the father?
  8. 8. Methods ● First, both men must swish with saline and spit it into a cup, which will be centrifuged and treated with Chelex. It will then be heated and centrifuged again. This isolates DNA. ● The DNA must then be amplified using PCR. ○ Primer/ddH2O mix and Ready-to-Go PCR Bead ○ Put through 30 cycles of PCR to amplify DNA ■ Each cycle includes initial denaturing at 94C for 2 minutes, denaturing at 94C for 30 seconds, annealing at 58C for 30 seconds, extending at 72C for 6 minutes. After the 30 cycles, the final extension will be at 72C for 6 minutes, followed by an indefinite hold at 4C.
  9. 9. ● The mother and child must both complete this process as well. ● The samples will all undergo gel electrophoresis to search for the presence of microsatellites at Chromosomes 5 and 9. ○ Agarose gel electrophoresis is the best method because it is effective in separating DNA, and is cheap and easy to perform. ● PEAK SCANNER will then be used to analyze the fragments and find the length of the microsatellite region, effectively genotyping them ○ The repeat region for each was 4 bases
  10. 10. ● D5S818 had twelve alleles: ○ 163, 155, 140, 139, 136, 135, 132, 131, 128, 124, 123, 115 ● D9S934 had nine alleles ○ 226, 222, 218, 216, 214, 210, 208, 206, 202 ● To calculate the number of possible genotypes, take the factorial of the number of alleles ○ 12! = 78 ■ Observed: 17/78 = 21.79% ○ 9! = 44 ■ Observed: 19/45 = 42.22% ● To account for both loci, multiply these values together ○ 78*44 = 3,510 ○ Only 37 of these were observed (1.05%)
  11. 11. Is This Population in Hardy-Weinberg Equilibrium? ● For Chromosome 5: Table 1: Allelic Frequencies of Chromosome 5 p q r s t u v w x y z a Allele 163 155 140 139 136 135 132 131 128 124 123 115 Freq 2/92 5/92 1/92 13/92 29/92 5/92 31/92 2/92 4/92 2/92 1/92 1/92 Freq .022 .054 .011 .207 .315 .054 .337 .022 .043 .022 .011 .011
  12. 12. ● For Chromosome 9: Table 2: Allelic Frequencies of Chromosome 9 p q r s t u v w x Allele 226 222 218 216 214 210 208 206 202 Freq 3/92 5/92 10/92 1/92 27/92 28/92 6/92 12/92 4/92 Frequ .033 .054 .109 .011 .293 .304 .065 .130 .043
  13. 13. Discussion ● Which Alleles were most and least common at each loci? ○ Most common alleles on Chromosome 5: 136 and 132 with their respective allelic frequencies being .326 and .315. ○ Least common alleles on Chromosome 5: 140, 123, and 115 all with allelic frequencies of .011, as each was only present once. ○ Most common alleles on Chromosome 9: 214, and 210 which their respective allelic frequencies being .293 and .283. ○ Least common alleles on Chromosome 9: 216 is the least common allele, with an allelic frequency of .011.
  14. 14. Discussion ● Genotypes observed at each loci: ○ 17 genotypes observed on chromosome 5 ○ 18 genotypes observed on chromosome 9 ● Up to 13 samples shared the same genotype for one locus however there were no samples with the same genotypes for both loci. ● Extremely unlikely that two members of a group of 46 would have the same two genotypes given there are 3,510 possible combinations.
  15. 15. Discussion ● In reality, two loci would not hold enough information to make a confident conclusion, generally many more loci are tested to make a diagnosis or determine paternity. ● It can be seen in the frequency of the alleles seen in the results section that it is actually significantly probable to share the same alleles with another person at one loci. This probability decreases with each additional loci examined.
  16. 16. Discussion Genotype for mother (S3-3) : 132/135/218/218 Genotype for child (S3-5): 135/136/218/214 Genotype for S3-4 : 132/136/210/214 Genotype for S3-6: 136/139/208/216 ● Question: Is S3-4 or S3-6 the father of S3-5? ○ Conclusion: S3-4 must be the father of S3-5. This is because the child has 136, 214, and 218 alleles which can only be inherited from the father. The only possible father with all three of these alleles is S3-4. ○ While this is an appropriate conclusion given our data based on two loci, this again would not be enough evidence to prove paternity.
  17. 17. Sources SLIDE 4 Brandt-Casadevall, C., Gené, M., Borrego, N., Gehrig, C., Dimo-Simonin, N., & Mangin, P. (2003, September 25). International Congress Series. Download PDFs. Retrieved April 16, 2015, from http://www.sciencedirect.com/science/article/pii/S0531513103010525. El-Alfy, S., & Abd El-Hafez, A. (2012, June 14). Paternity testing and forensic DNA typing by multiplex STR analysis using ABI PRISM 310 Genetic Analyzer. Journal of Engineering and Biotechnology. Retrieved April 19, 2015, from http://www.sciencedirect. com/science/article/pii/S1687157X12000194. SLIDES 5 & 6 Tozaki, T., Kakoi, H., Mashima, S., Hirota, K., Hasegawa, T., Ishida, N., . . . Tomita, M. (n.d.). The Journal of Veterinary Medicine. Retrieved April 23, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/11767052. DISCUSSION Sakaoka, K., Suzuki, I., Kasugai, N., Fukumoto, Y. (2014). Zoo Biology. Retrieved April 23, 2015, from http://www.ncbi.nlm.nih. gov/pubmed/25157452. Isberg, S., Chen, Y., Barker, S., Moran, C. (2004). The Journal of Heredity. Retrieved April 23, 2015, from http://www.ncbi.nlm.nih.gov/pubmed/? term=microsatellite+paternity+testing+crocodiles.

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