Isolation and cloning of DNA from somatic tissue of soft corals (Cnidaria: Octocorallia)

Abstract

A method is described for isolating high molecular weight D N A from somatic tissue of soft corals. The method avoids problems associated with the presence of nucleases, pigments and other secondary metabolites in soft corals. Tissue is frozen in liquid nitrogen, pulverized with a pestle and mortar, and the powder extracted with buffered sodium dodecyl sulphate at 4 °C in the presence of phenol/chloroform. This technique has been applied to Alcyonium, Sinularia, Sarcophyton and Lobophytum species. Repeated sequences have been cloned from Alcyonium sp., and used to probe slot-blots of genomic D N A and Southern blots of restriction digests. Homologous repeated sequences were detected in three Alcyonium sp., but not in three Sinularia sp., despite these genera being closely related.

Reducing host DNA contamination in 16S rRNA gene surveys of anthozoan microbiomes using PNA clamps

“…Samples from P. panamensis yielded low DNA concentrations, which may suggest that P. panamensis cells did not lyse as readily as gorgonian cells, leading to a sample composed mainly of symbiotic microbial DNA. Lysis affinity of cells varies based on cell size, the amount of mesoglea, and the presence of inhibitors (ten Lohuis et al 1990;Weber et al 2017). The disparity in initial contamination may also have been due to a higher number of mismatches between P. panamensis mtDNA and the microbial primers used, reducing the amplification of the coral DNA.…”

“…Traditional microbial cell lysis methods can lyse coral cells, swamping samples with eukaryotic DNA (Galkiewicz and Kellogg 2008;Weber et al 2017). Additionally, corals cells are also rife with heavy pigmentation and nucleases, both of which are PCR inhibitors (ten Lohuis et al 1990; Price and Linge 1999). These issues are problematic for coral microbiome studies because coral DNA is easily amplified by the bacterial specific primers employed by large-scale microbial sequencing projects (e.g., Earth Microbiome Project; Galkiewicz and Kellogg 2008;Weber et al 2017).…”

Immunological evidence for a ribulose bisphosphate carboxylase-like protein in the symbiotic dinoflagellate Symbiodinium sp.

“…Symbiodinium sp. was isolated from the hard coral Acroporaformosa by the Waterpik technique [4], from the soft coral Smularia by gentle compression [5] and from clam (Tridacna gigas) mantle tissue essentially as described by Griffiths and Streamer [6]; the isolates were washed extensively (> 10 x ) and the purity and integrity of algal preparations confirmed by optical microscopy prior to lysis. Cultures of Symbioditlium isolated form the anemone Aiptasia pulchella (API92) and the clam Tridacna gigas (TG169) were obtained from Dr. W. Fitt (University of Georgia); culture conditions were as previously described [7].…”