30DNA from formalin-preserved tissue could unlock a vast repository of genetic information stored in 31 museums worldwide. However, formaldehyde crosslinks proteins and DNA, and prevents ready 32 amplification and DNA sequencing. Formaldehyde acylation also fragments the DNA. Treatment with 33 proteinase K proteolyzes crosslinked proteins to rescue the DNA, though the process is quite slow. To 34 reduce processing time and improve rescue efficiency, we applied the mechanical energy of a vortex 35 fluidic device (VFD) to drive the catalytic activity of proteinase K and recover DNA from American lobster 36 tissue (Homarus americanus) fixed in 3.7% formalin for >1-year. A scan of VFD rotational speeds 37 identified the optimal rotational speed for recovery of PCR-amplifiable DNA and while 500+ base pairs 38 were sequenced, shorter read lengths were more consistently obtained. This VFD-based method also 39 effectively recovered DNA from formalin-preserved samples. The results provide a roadmap for exploring 40 DNA from millions of historical and even extinct species.
42Archived biological samples offer an important source of genetic information for diverse fields including 43 evolutionary biology, ecology, phylogenetics, biodiversity, and epidemiology [1-2]. Samples, from 44 hydrated tissues to whole organisms, have historically been preserved in aqueous formaldehyde (3.7 to 45 4% solution of formaldehyde in water, termed formalin). In many cases, these specimens are the only 46 remaining samples that could provide genetic information about the organisms, including their 47 microbiomes, environments, diets, and other attributes -all from the moment of sample preservation [3-48 5]. This preservative, however, hinders DNA amplification and sequencing with the sample [6]. Thus, new 49 methods to recover DNA from formalin-fixed specimens could advance our ability to access the genetic 50 information in these samples, and advance our understanding of how organisms and ecosystems have 51 responded to natural and anthropogenic changes over time. For example, formalin-fixed specimens in 52 natural history museums could be used to elucidate the impact of environmental changes on the DNA of 53 biological populations [1-2, 7]. DNA sequencing of such samples could address longitudinal, biological 54 questions that may be impractical to address without the genetic information for the preserved specimens 55 [2, 8-9].56 For >150 years, formalin fixation has been used to effectively preserve hydrated specimens [7]. A 57 vast repository of formalin-fixed samples exists, including at least 400 million samples at 13 large 58 institutions [1]. Marine organisms are particularly well-preserved in this aqueous preservative, as it retains 59 morphological features well, enabling more detailed taxonomic studies. Aqueous formaldehyde is also 60 advantageous in that it stops parasitic microbial growth [10]. However, preserving samples in formalin 61 fixation damages DNA [11-12]. Covalent modification of DNA bases by the electrophilic formaldeh...
