DESS deconstructed: Is EDTA solely responsible for protection of high molecular weight DNA in this common tissue preservative?

Abstract

DESS is a formulation widely used to preserve DNA in biological tissue samples. Although it contains three ingredients, dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA) and sodium chloride (NaCl), it is frequently referred to as a DMSO-based preservative. The effectiveness of DESS has been confirmed for a variety of taxa and tissues, however, to our knowledge, the contributions of each component of DESS to DNA preservation have not been evaluated. To address this question, we stored tissues of three aquatic taxa, Mytilus edulis (blue mussel), Faxonius virilis (virile crayfish) and Alitta virens (clam worm) in DESS, each component of DESS individually and solutions containing all combinations of two components of DESS. After storage at room temperature for intervals ranging from one day to six months, we extracted DNA from each tissue and measured the percentage of high molecular weight (HMW) DNA recovered (%R) and normalized HMW DNA yield (nY). Here, HMW DNA is defined as fragments >10 kb. For comparison, we also measured the % R and nY of HMW DNA from extracts of fresh tissues and those stored in 95% EtOH over the same time intervals. We found that in cases where DESS performed most effectively (yielding � 20%R of HMW DNA), all solutions containing EDTA were as or more effective than DESS. Conversely, in cases where DESS performed more poorly, none of the six DESS-variant storage solutions provided better protection of HMW DNA than DESS. Moreover, for all taxa and storage intervals longer than one day, tissues stored in solutions containing DMSO alone, NaCl alone or DMSO and NaCl in combination resulted in %R and nY of HMW DNA significantly lower than those of fresh tissues. These results indicate that for the taxa, solutions and time intervals examined, only EDTA contributed directly to preservation of high molecular weight DNA.

Extension of Mitogenome Enrichment Based on Single Long-Range PCR: mtDNAs and Putative Mitochondrial-Derived Peptides of Five Rodent Hibernators

“…The assay for mitogenome amplification applicable for fresh or well-preserved blood and tissue samples can be helpful to assess issues of mitogenomics such as point and length heteroplasmy of clinical and forensic samples ( Cagnin et al, 2009 ), evolutionary mechanisms of heteroplasmy ( Tikochinski et al, 2020 ) or intraspecific phylogeography ( Avise et al, 1987 ). Our assay holds promise for tissue samples preserved in ethylenediaminetetraacetic acid (EDTA) ( Sharpe et al, 2020 ), or choline-based ionic liquids ( Oosting et al, 2020 ) as well as for certain applications based on aquatic eDNA ( Deiner et al, 2017 ; Ding et al, 2020 ). The proof that eDNA derived from macro-organisms inhabiting a water body remains intact at least at the size of vertebrate mitogenomes (∼16 kb) contradicts the former common view that eDNA from communities of living fishes in water is highly degraded ( Bohmann et al, 2014 ).…”

Contrasting Modes of Mitochondrial Genome Evolution in Sister Taxa of Wood-Eating Marine Bivalves (Teredinidae and Xylophagaidae)

“…For Xylophagaidae, siphon tissue was dissected from each bivalve, and total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocols. Tissue samples collected in Alabama and Florida were preserved in 0.25 M EDTA, pH 8.0 ( Sharpe et al 2020 ). Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) and concentrated using the DNA Clean & Concentrator-25 Kit (Zymo Research) following manufacturer’s recommended protocols.…”

Opportunities and challenges for high‐quality biodiversity tissue archives in the age of long‐read sequencing

“…DMSO is frequently used in combination with a saturated salt concentration to prevent enzymatic denaturation and a chelator such as EDTA that prevents oxidative damage (DESS). The effectiveness of DESS has been confirmed for a variety of taxa and tissues (Sharpe et al, 2020), and is perhaps one of the most promising preservatives when snap freezing samples in liquid nitrogen is not an option. However, further research is urgently needed to optimize taxa‐specific protocols and quantify the efficacy of liquid preservatives relative to cryopreservation.…”

“…The production of hyxdroxyl radicals via Fenton type reactions is dependent on the presence of trace metals (Molina & Anchordoquy, 2007) and chelating agents can be used to bind free metals. EDTA is commonly used as a chelating agent (Sharpe et al, 2020) and is an effective preservative when combined with a radical scavenger (Anchordoquy & Molina, 2007; Klingström et al, 2018). Queen Lysis buffer for example, which includes EDTA, is frequently used to store unfrozen blood samples (Seutin et al, 1991) from species with nucleated red blood cells and has even resulted in better genome assemblies than DNA retrieved from museum‐grade frozen tissues (Bakker et al, 2020).…”