DNA extraction from formalin-fixed tissue: new light from the deep sea

Abstract

SUMMARY: DNA samples were extracted from ethanol and formalin-fixed decapod crustacean tissue using a new method based on Tetramethylsilane (TMS)-Chelex. It is shown that neither an indigestible matrix of cross-linked protein nor soluble PCR inhibitors impede PCR success when dealing with formalin-fixed material. Instead, amplification success from formalin-fixed tissue appears to depend on the presence of unmodified DNA in the extracted sample. A staining method that facilitates the targeting of samples with a high content of unmodified DNA is provided.Keywords: tetramethylsilane, ethidium bromide, formalin, Carcinus, Lithodidae. RESUMEN: Extracción de ADN a partir de tejido fijado en formol: nueva luz desde el mar abisal. -Muestras de ADN de distintos crustáceos decápodos fueron obtenidas independientemente a partir de tejidos fijados en etanol y tejidos fijados en formol mediante un nuevo protocolo basado en el Tetrametilsilano (TMS)-Chelex. Los resultados obtenidos muestran que el ADN no se encuentra atrapado de forma irreversible en una matriz proteica y que el éxito de amplificación no depende de la extracción de inhibidores de PCR solubles. Sin embargo, nuestros resultados indican que el éxito de amplificación depende de la presencia de ADN no modificado en la muestra. Se incluye un sencillo método de tinción que facilita la identificación de muestras con un alto contenido en ADN no modificado.Palabras clave: tetrametilsilano, bromuro de etidio, formol, Carcinus, Lithodidae.

Following the Phoenician example: western Mediterranean colonization by <em>Spirobranchus</em> cf. <em>tetraceros</em> (Annelida: Serpulidae)

“…Total genomic DNA was extracted from samples of Spirobranchus collected from Heraklion, Valencia and NSW (Australia) (see Table 1 for details) using a QIAamp DNA Mini Kit (QIAGEN Inc) and following the manufacturer's instructions. DNA quality was assessed by gel electrophoresis (1% agarose) (Palero et al 2010) and quantified using a Qubit 3.0 fluorometer (Life Technologies). A fragment (~400 bp) of the mitochondrial cytochrome b gene was amplified with ~30 ng of genomic DNA in a reaction containing 1 U of Taq polymerase (Amersham), 1 × buffer (Amersham), 0.2 mM of each primer (Cytb 424F = GGWTAYGT-WYTWCCWTGRGGWCARAT and Cytb 876R = GCRTAWGCRAAWARRAARTAYCAYTCWGG; Boore and Brown (2000)) and 0.12 mM dNTPs.…”

Access to Marine Genetic Resources (MGR): Raising Awareness of Best-Practice Through a New Agreement for Biodiversity Beyond National Jurisdiction (BBNJ)

“…These could include sea water, sea ice or sediment samples from ABNJ, collected for environmental chemistry or physical oceanography research. While most utilisation of MGR will come from live cultured, frozen or ethanol-preserved materials, sequencing technologies are advancing very rapidly, and extraction of DNA from formalin-fixed and ancient materials is now an established practice (Palero et al, 2010;Cook et al, 2015;Ruane and Austin, 2017). Therefore, MGR could also be obtained from samples neither originally collected nor preserved for molecular work.…”

Larval stages of the deep-sea lobster Polycheles typhlops (Decapoda, Polychelida) identified by DNA analysis: morphology, systematic, distribution and ecology

“…Total genomic DNA extraction from the first zoea specimen captured during supra-benthos sampling south of the Balearic Sea (Station: 39.067N-2.675E; Table 1) and the adult specimens from Mediterranean waters was performed using the Chelex-resin method (Palero et al 2010). The standard universal primers for DNA barcoding (Folmer et al 1994) were used for PCR amplification, given that they had been previously tested in Polycheles with positive results (see Palero et al 2009).…”