- 1. Sex Determination of Juvenileand Uknown Maya SkeletonsMackenzie Kilkeary
2. Statement of Hypotheses If there is an amplifiable amount ofancient DNA (aDNA) in a single tooth of aMaya skeleton, then the sex of theindividual skeleton can be determined. If the sex of unknown and juvenileskeletons can be determined, then abetter understanding of ancient Mayaculture can be reached. 3. Research Goals Determine if the DNA amplification protocolworks on human saliva samples beforeworking on ancient teeth. Find the annealing temperature that resultsin clearest bands during gel electrophoresis. Determine the sex of as many individualskeletons as possible targeting theAmelogenin gene. 4. Amelogenin Gene Found on both the X and Y Chromosome. Codes for a major protein involved in thedevelopment of enamel. Differs in size on X and Y chromosome. Different reverse primers are used forAmelogenin genes on X and Ychromosomes. 5. Primers for the Amelogenin Gene Primers for the Amelogenin gene consist of a forward primer for both X andY, as well as a reverse primer specific for X and for Y. Table 2. Information on primers used for sex identification (Stone et al., 1996) FragmentSiteSequence Tm Size (basepairs) Amelogenin GeneF 5-X and Y GCTCATATTATACTT 51.2C - Chromosome GACAAAGCA-3R 5- Amelogenin GeneGACGTCGGGTTTGA 57.8C191X Chromosome GGTTCTC-3R 5- Amelogenin GeneAGGTAAAATTACTAA 52.6C 157Y ChromosomeCTTTGGGCA-3 6. Preliminary Methods: Testing Human DNA To test and perfect protocol, human saliva samples were used. Saliva samples were retrieved from colleagues and students in Biology I Laboratory. Samples were boiled in 100l of Chelex to burst cells and cooled in ice. Samples are spun down, leaving unwanted debris in pellet, and 30l of supernatant is removed (contains DNA). 7. Preliminary Methods: Testing Human DNA DNA Amplification was accomplished by adding 2l of DNA, 2.5l of forward primer, 2.5l of reverse primer and 18l of dH2O to a PCR tube containing a Ready-To-Go Bead. 2l DNA + 2.5l FP + 2.5l RP + 18l dH2O = 25l Two PCR tubes are prepared for each sample. One for the X chromosome and Y chromosome. Different reverse primers used for X and Y. Polymerase Chain Reaction contains three steps: (1) Denaturing (2) Annealing (3) Extension. 8. Preliminary Methods: Testing Human DNA Denaturing pulls the double-stranded DNA apart. Annealing allows the primers to stick to corresponding sequences on the DNA. Extension gives the Taq polymerase an opportunity to extend bases off of primers, complementary to corresponding strand, to complete the DNA sequence we are looking for. Annealing temperatures were varied to determine which temp resulted in the clearest bands. 9. Annealing Temperatures: 52.5C to 60CTable 1. Polymerase Chain Reaction Settings for Male 2 and6s Saliva Samples ExtensionIndividual Denaturing Step Annealing StepStepo oo Male 2 30 sec @ 94 C1 min @ 60 C 1 min @ 72 Co oo Male 2 30 sec @ 94 C 1 min @ 59.6 C 1 min @ 72 Co oo Male 2 30 sec @ 94 C 1 min @ 58.8 C 1 min @ 72 Co oo Male 2 30 sec @ 94 C 1 min @ 58.2 C 1 min @ 72 Co oo Male 6 30 sec @ 94 C1 min @ 58 C 1 min @ 72 Co oo Male 6 30 sec @ 94 C 1 min @ 56.8 C 1 min @ 72 Co oo Male 6 30 sec @ 94 C 1 min @ 54.4 C 1 min @ 72 Co oo Male 6 30 sec @ 94 C 1 min @ 52.5 C 1 min @ 72 C 10. Preliminary Methods: Testing Human DNA PCR results were analyzed using gel electrophoresis and gel imaging. 100 base pair molecular ladder used for gel. 4l of loading dye added to each sample. Bands: looking for X band at ~191 base pairs and a Y band at ~157 base pairs. 11. Preliminary Results: Testing Human DNA Annealing temperatures from 52.5C to 58C for Male Sample 6 (inverted).Band Band~191 ~157 12. Preliminary Results: Testing Human DNA Annealing temperatures from 58.2C to 60C for Male Sample 2. Band ~191Band ~157 13. Preliminary Results: Testing Human DNA If a sample has a band at ~191 for X and ~157 for Y then the sample is identified as male (males have X and Y chromosome). If a sample has a band at ~191 for X and is lacking a band ~157 for Y then the sample may be identified as female (females have two X chromosomes). Although difficult to see, an annealing temperature at 60C warranted a clear band for X and Y, while alleviating most unwanted bands. Another four human saliva samples were tested with a 60C annealing temperature for verification 14. Preliminary Results: Testing Human DNA Samples at 60C: Female 02, Female 01, Male 03 and Male 01.NoBandBand Band Band~191~157 ~191 ~157 15. Methods: Ancient DNA Extraction Ancient Maya tooth sample is first sterilizedby scrubbing the tooth with 10% bleach andtoothbrush followed by ultra violet radiationfor 1 hour. The tooth is then frozen overnight. The tooth is crushed using a mortar andpestle to a fine powder and material inplaced in a 1.5ml sterile tube. 16. Methods: Ancient DNA Extraction Tooth material is incubated at between 45-60C in DeHybernation A Solution for 2 to12 hours. The solution is centrifuged at high speed forfive minutes and supernatant is transferredto new centrifuge tube. Room incubation of supernatant with DNAGLASSMILK between 10 and 30 minutes. 17. Methods: DNA Isolation and Amplification Using a spin filter and catch tube, the solution undergoes a series of washes to isolate any DNA. DNA amplification protocol is the same as used for human saliva samples except for the amount of DNA (aDNA) used. 8l DNA + 2.5l FP + 2.5l RP + 12l dH2O = 25l 18. Results: Unknown Maya Skeleton 432 Maya 432: Identified as a female. X band present at ~191 base pairs and no Y band present ~157 base pairs. Band at 191bp No Band at 157bp100 bp ladder X Y 19. Ancient DNA Analysis Thus far, a unknown male (Maya 442)and female skeleton (Maya 432) havebeen identified. The gel for the male wasextremely difficult to read, however DNAsequencing confirmed the results. Obtaining a Male identification increasesthe validity of the female identificationbecause a female identification relies ona negative result for the Y chromosome. 20. Difficulties with aDNA Ancient DNA is typically several years old and has endured varying environmental conditions. Many time aDNA can undergo degradation and fragmentation over its lifetime, so obtaining results can be difficult. The researcher has extracted/isolated/amplified close to 20 separate tooth samples and only received two results. Contamination is also a huge worry for any researcher working with aDNA. A sterile laboratory and working conditions were ensured before DNA extraction and isolation was taken place. 21. Conclusions/Research Summary DNA amplification protocol and amelogenin primerswere tested on human saliva samples first and anappropriate annealing temperature was found. DNA Extraction, Isolation and Amplification protocolsyielded two positive results (Maya 442, Maya 432)from skeletal collection thus far, one male andfemale. Results of previously unknown skeletons are onestep closer to better understanding the burialpatterns and culture of Maya population in Tipu,Belize. 22. Acknowledgements The researcher is pleased to thank Dr. MarkCohen for allowing access to the Mayaskeleton SUNY Plattsburgh bone laboratory. Another special thanks to Dr. Nancy Elwessand Mrs. Sandra Latourelle for theirpatience and willingness to help in everyway possible. The researcher would also like to thankEdmund Adjapong for the assistance withDNA extraction.