Optimization of DNA extraction from a scleractinian coral for the detection of thymine dimers by immunoassay.
Ultraviolet (UV)-B is known to cause DNA damage, principally by the formation of thymine dimers, but little research has been conducted in coral reef environments where UV doses are high. The majority of tropical reef-dwelling corals form a mutualistic symbiosis with the dinoflagellate Symbiodinium but few studies have been conducted on in situ DNA damage in corals and none have investigated the symbiotic components separately. The aim of this research was to quantify DNA damage in both the coral host and the dinoflagellate symbiont. The first step in this investigation was to optimize the extraction of DNA from the host, Porites astreoides, as well as the symbiont. The optimization was divided into a series of steps: the preservation of the samples, separation of the coral tissue from the skeleton, separation of the host tissue from the algal cells to prevent cross contamination as well as the extraction and purification of genomic DNA from the algae that are located intracellularly within the invertebrate animal tissue. The best preservation method was freezing at low temperatures without ethanol. After scraping with a razor blade, the coral tissue can be divided into host and algal components and the DNA extracted using modifications of published techniques yielding DNA suitable for the quantification of thymine dimer formation using antibodies. Preliminary data suggest that in P. astreoides collected from 1 m depth, thymine dimers form approximately 2.8 times more frequently in the host DNA than in the DNA of its symbionts.
Banaszak
Anastazia T
AT
Photobiology Laboratory, Unidad Académica Puerto Morelos, Instituto de Ciencias del Mar y Limnología, Universidad Nacional Autónoma de México, Puerto Morelos, Mexico. banaszak@mar.icmyl.unam.mx
D000818:Animals; D037421:Anthozoa / Q000235:genetics*; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D019281:Dimerization; D007118:Immunoassay / Q000379:methods*; D013941:Thymine / Q000737:chemistry*
D004247:DNA; D013941:Thymine