Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.
Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species - a notion that was long-debated without the benefit of this new and compelling evidence.
19Current molecular biology laboratories rely heavily on the purification and manipulation of 20 nucleic acids. Yet, commonly used centrifuge-and column-based protocols require 21 specialised equipment, often use toxic reagents and are not economically scalable or practical 22 to use in a high-throughput manner. Although it has been known for some time that magnetic 23 beads can provide an elegant answer to these issues, the development of open-source 24 protocols based on beads has been limited. In this article, we provide step-by-step 25 instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols 26 for their use in the high-throughput purification of plasmids, genomic DNA and total RNA from 27 different sources, as well as environmental TNA and PCR amplicons. We also provide a bead-28 based protocol for bisulfite conversion, and size selection of DNA and RNA fragments. 29Comparison to other methods highlights the capability, versatility and extreme cost-30 effectiveness of using magnetic beads. These open source protocols and the associated 31 webpage (https://bomb.bio) can serve as a platform for further protocol customisation and 32 community engagement. 33 3 Abbreviations 34 BOMB: Bio-On-Magnetic-Beads 35 SPRI: Solid-Phase Reversible Immobilisation 36 MNP: magnetic nanoparticle 37 38The authors would like to thank all members of the Jurkowski and Hore laboratories for 456helping to optimise and test the BOMB protocols. We are also indebted to Ken Wyber (Otago 457Polytechnic) for help with laser cutting magnetic plates. We are grateful to Dr. Renata 458Jurkowska for critical reading of the manuscript. We would like to thank the wider research 459 community for offering unpublished information and resources concerning magnetic bead 460 22 preparation and utility, in particular, Dr Ethan Ford, Dr James Hadfield, Dr Brant Faircloth, Dr 461 Nadin Rohland and associated authors. 462 Author's contribution 463 The idea was conceived by TPJ and TH. Protocol setup and optimisation was led by PO, PS, 464 DB, TPJ and TH, with contributions from SH, JF, VM, LS, VJS, G-JJ and FvM. Laser cutting 465 designs were contributed by SRH. The electron microscope analysis was done by KH. The 466 website and its content were created by TM, PS, PO, TPJ and TH. The manuscript was written 467 by TPJ, TH, PS and PO. All authors contributed to the editing of the manuscript and approved 468 its final version. 469
Fish show extraordinary sexual plasticity, changing sex naturally as part of their life cycle or reversing sex because of environmental stressors. This plasticity shows that sexual fate is not an irreversible process but the result of an ongoing tug-of-war for supremacy between male and female signaling networks. The behavioral, gonadal, and morphological changes involved in this process are well described, yet the molecular events that underpin those changes remain poorly understood. Epigenetic modifications emerge as a critical link between environmental stimuli, the onset of sex change, and subsequent maintenance of sexual phenotype. Here we synthesize current knowledge of sex change, focusing on the genetic and epigenetic processes that are likely involved in the initiation and regulation of sex change. We anticipate that better understanding of sex change in fish will shed new light on sex determination and development in vertebrates and on how environmental perturbations affect sexual fate.
In mammals, females generally live longer than males. Nevertheless, the mechanisms underpinning sex-dependent longevity are currently unclear. Epigenetic clocks are powerful biological biomarkers capable of precisely estimating chronological age and identifying novel factors influencing the aging rate using only DNA methylation data. In this study, we developed the first epigenetic clock for domesticated sheep (Ovis aries), which can predict chronological age with a median absolute error of 5.1 months. We have discovered that castrated male sheep have a decelerated aging rate compared to intact males, mediated at least in part by the removal of androgens. Furthermore, we identified several androgen-sensitive CpG dinucleotides that become progressively hypomethylated with age in intact males, but remain stable in castrated males and females. Comparable sex-specific methylation differences in MKLN1 also exist in bat skin and a range of mouse tissues that have high androgen receptor expression, indicating it may drive androgen-dependent hypomethylation in divergent mammalian species. In characterising these sites, we identify biologically plausible mechanisms explaining how androgens drive male-accelerated aging.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.