Bachelor's Essays (Embargoed)

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Application of Methylation-Sensitive Amplified Fragment Length Polymorphism Analysis to the Loggerhead Sea Turtle, Caretta caretta, and the American Alligator, Alligator mississippiensis
King, Katharine R; Shedlock, Andrew M
Methylation-sensitive Amplified Fragment Length Polymorphism genotyping was used to investigate alligator and turtle genomes in an effort to evaluate differential DNA methylation by sex. Genomic DNA extracted from whole blood was digested with EcoRI and either HpaII or MspI, isoschizomers with differing sensitivity to methylation. Fluorescently labeled primers plus three selective nucleotides were used to amplify a subset of the total fragment pool. Fragments were visualized and scored using the Beckman CEQ capillary electrophoresis system. Statistical analysis showed a significant difference between male and female samples in overall methylation for one of the three primer pairs tested on the alligator genome with males having higher levels of methylation (84%) than females (57%; p < 0.003). Our proof-of-principle results demonstrate that MS-AFLP complements standard AFLP diagnostics and can be integrated cost-effectively with indexed high-throughput DNA sequencing strategies for investigating methylation-sensitive mechanisms of gene control in temperature-dependent sex determining organisms.