Current Protocols in Nucleic Acid Chemistry


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Initial purification can be carried out from a variety of sources, which commonly include bacterial cultures and plant and animal cells or tissues. Nucleic acids may also be derived from cell-free sources, such as blood plasma, various environmental substrates, or from in vitro reactions like PCR. Once purified, nucleic acids are usually manipulated in some manner ahead of analysis or functional use—a process that in turn requires further appropriate reagents and handling.

Most current nucleic acid purification and manipulation techniques rely upon either a commercially produced silica-based column or a centrifuge often both. In addition to commonly using toxic chemicals such as phenol, these protocols are generally not suitable for high-throughput approaches, whereby 96 or more samples are processed simultaneously.

This is because standard benchtop centrifuges only hold 24 tubes and multichannel pipettes or liquid handling robots cannot be used to accelerate the isolations. Moreover, the per-sample cost of silica columns make processing large numbers prohibitively expensive, and other available commercial high-throughput solutions for nucleic acids extractions are still not affordable for most molecular biology laboratories.

Magnetic beads are small nano- or microparticles and have long been recognised as a way to solve scalability issues with respect to nucleic acid purification and manipulation [ 2 — 4 ].

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Their most useful characteristic is the ability to achieve solid-phase reversible immobilisation SPRI [ 3 ], meaning they can reversibly bind nucleic acid under dehydrating conditions and, when in the presence of a strong magnet, can be safely immobilised throughout multiple wash and manipulation steps.

Magnetic bead protocols are inherently scalable due to the fact that they are independent of centrifugation and the required materials are exceedingly cheap both to purchase and manufacture in a laboratory setting. However, despite these attractive attributes, surprisingly little community effort has been committed to the development of open-source protocols featuring their use. Here, we present Bio-On-Magnetic-Beads BOMB , an open-source platform consisting of both novel and existing magnetic bead-based protocols that are capable of a wide-range of nucleic acid purification and manipulation experiments Fig 1.

We first detail a method for simple synthesis of magnetic nanoparticles MNPs and their functionalisation with either a silica or carboxyl coating that can be performed in any molecular biology laboratory with standard equipment. We further show how cheap magnetic immobilisation devices can be assembled or fabricated for 1. We have also developed an open-source protocol for bisulfite conversion of DNA used in epigenetic analysis and update existing protocols for size selection of DNA fragments. The circled numbers indicate the protocols for the respective procedure.

Given the impressive economic advantages of magnetic beads for nucleic acid extraction and manipulation, both in terms of capital outlay and per-sample costs, we consider the BOMB platform a positive step towards the democratisation of life sciences. The essential components of a magnetic bead platform are the beads themselves and a magnet strong enough to immobilise them.

Although many life science researchers will be familiar with proprietary beads e. However, in order to do so, some potentially unfamiliar concepts need to be explained. Both bead types work well for nucleic acid purification and manipulation; however, their different physical and chemical properties do change their behaviour.

For example, the polymer within the larger ferrite-polymer beads effectively lowers the density of the bead so they are less likely to settle out of the suspension during handling steps. The smaller solid-core ferrite beads have a larger relative surface area for binding and can also be easily made in a standard molecular biology laboratory see BOMB protocols 1—3.

A key aspect of magnetic beads used for molecular biology is that, irrespective of their size, they need to be chemically coated. The first reason for doing this is to provide stability for the bead. Without coating, oxidation of the ferrite would lead to contamination of potentially sensitive samples with iron ions, and the beads would lose their magnetic properties over time. In addition, chemical coating grants additional function to magnetic beads. For example, silica- or carboxyl-polymer coatings are most commonly used because, in addition to providing bead stability, they are relatively chemically inert silica or negatively charged carboxyl , thus facilitating desorption of the negatively charged nucleic acids from the beads during elution steps.

Here, we outline a simple protocol for preparation of silica- or carboxyl-coated beads in a standard life science laboratory and production of magnetic racks suitable for their immobilisation. Ferrite nanoparticles can be synthesised using various protocols reviewed in [ 6 , 7 ].

Current protocols in nucleic acid chemistry (Online) [2000]

We adopted the broadly used coprecipitation method due to its simplicity and efficiency, but also because it does not require any specialised equipment [ 8 ]. The ferrite particles synthesised using this approach have a diameter of approximately 5 to 20 nm as judged by transmission electron microscopy TEM images S1A Fig.

After synthesis, the core particles are extensively washed with deionised water. In order to prevent oxidation of the ferrite, we recommend coating the beads immediately after synthesis. However, it is also possible to stabilise them in the short term using detergents, sodium oleate, polyvinylpyrrolidone PVP , or other chemicals reviewed in [ 6 , 7 ] , and upon lyophilisation, they can be stored in an air-tight container under inert atmosphere.

In a similar fashion to storing solutions inside glass bottles, encasing ferrite nanoparticles in silica prevents magnetic bead oxidation and leakage of iron ions. Silica coating also provides an inert surface for precipitation of nucleic acid without the risk of irreversible association. The thickness of the silica coat and therefore the size of the particle can be controlled through the addition of an increasing amount of TEOS [ 10 ].

nucleic acid chemistry

The provided standard coating protocol results in the silica-coated beads with a size of approximately nm S1B Fig , which perform well for a wide range of the nucleic acid purification and manipulation experiments S1 Appendix , BOMB protocol 2. An alternative way to stabilise magnetic particles is to coat them with carboxyl-modified polymer S1C Fig. Although potentially not providing the same stability as silica, carboxyl coating endows the ferrite core with a weak negative charge, thus altering its electrostatic interaction with nucleic acids and ultimately affecting bead functionality.

Although other reaction schemes are possible, we polymerise methacrylic acid monomers on top of the MNPs, thus providing a negatively charged carboxyl coat. For this, the ferrite core particles are dispersed with a detergent sodium dodecyl sulfate , and a layer of polymethacrylic acid PMAA is deposited on the surface of the beads by a free-radical retrograde precipitation polymerisation reaction [ 11 ] S1 Appendix , BOMB protocol 3. Although making your own magnetic beads is by far the most economical way to make use of magnetic beads for nucleic acid handling, for many laboratories, bead-based systems can still be developed relatively cheaply from commercial sources numerous beads are available; however, when used in protocols here, we purchased Sera-Mag SpeedBead Carboxylate Modified Magnetic Particles, Hydrophylic, 15 ml, cat.

A single bottle of carboxyl-functionalised beads can be purchased for approximately the same cost as a DNA extraction kit based on plastic silica columns for 50 to samples. Although there are advantages and disadvantages to either system, we find that our laboratory-made and commercially sourced beads show similar performance. MNPs are so useful because they can be immobilised and resuspended easily by moving them in and out of a magnetic field. The simplest way to do this is using immobilisation racks consisting of magnet arrays, either in microtube or microplate format.

Despite their simple construction and lack of moving parts, commercially available immobilisation racks are surprisingly expensive. However, they can be assembled in a laboratory setting relatively easily and cheaply. The most critical component of the rack is the magnet itself. In order to achieve rapid separation, it is best to use high-quality neodymium magnets, N42 grade or above. We have created a number of different racks that are suitable for specific vessel formats, using recycled materials or custom 3D-printed or laser-cut parts Fig 1.

For example, when using 8-strip PCR tubes, well microplates, and deep-well plates, ring magnets can be fixed onto the top of old pipette tip box components using cyanoacrylate adhesive e. For those who have access to laser cutting or 3D printing, we have designed well racks that are suitable for a range of deep-well plates and a 3D-printable plastic rack which can hold up to 8 microcentrifuge tubes S1 Appendix , BOMB protocol A. Once the essential BOMB tools i. Many experiments in molecular biology laboratories start with purification of nucleic acid from source cells or tissues Fig 2 and go on to perform some type of manipulation often involving many intermediate purification steps prior to final quantitation or introduction back into a biological system.

Magnetic bead-based protocols can mediate each of these purification and manipulation steps in both a universal and modular fashion Fig 3. This means that for almost any application, reliance upon purchased kits can be dramatically reduced or removed entirely. Here, we highlight some of the most commonly used protocols and their utility. Detailed protocols can be found in the Supporting Information. Different volumes of binding buffer compared to sample volume were used to achieve size exclusion.

Two volumes of cBB were used as a control relative to input. The volumes loaded are proportional i. MW: Hyperladder I Bioline. Underlying data for Fig. Purification of nucleic acid from a wide variety of biological and synthetic input sources left-hand panel can be performed using the BOMB platform.

Current Protocols in Nucleic Acid Chemistry

The separation and purification of nucleic acids following enzymatic reactions is a necessary procedure in a variety of biochemical assays and was originally performed by precipitation with salts and alcohol [ 12 — 14 ]. However, these methods usually require long incubation steps of up to several hours for smaller molecules and therefore have been surpassed by rapid methods involving commercial silica columns or SPRI on magnetic beads [ 3 ].

A major advantage of SPRI bead methods is the ability to perform sequential enzymatic clean-ups in one tube in an efficient manner [ 15 ], thus greatly simplifying complex nucleic acid handling procedures such as DNA library construction for next-generation sequencing [ 4 ]. Moreover, because larger fragments precipitate to magnetic beads more efficiently than smaller ones in hydrophilic conditions, bead immobilisation can be used to select or exclude nucleic acids of particular sizes by varying the binding conditions.

Whereas DNA is bound to carboxyl beads via molecular crowding with high concentrations of PEG and NaCl [ 16 ], binding DNA to silica beads utilises the altered affinity of the negatively charged DNA backbone to the silica surface in the presence of chaotropic salts [ 17 , 18 ]. We most commonly use silica-coated beads and guanidinium hydrochloride for capture. Furthermore, utilising the well format and the earlier described BOMB microplate racks, approximately samples can be processed within 45 minutes by a single person. Plasmid extraction from cultured Escherichia coli strains is probably one of the most common laboratory practices.

Numerous commercial kits are based on this technology, which employs either silica-packed columns or silica-coated magnetic beads. Both methods represent efficient and reliable techniques for DNA isolation with a reasonable cost of approximately 1. However, for processing samples in a high-throughput scale, the price can become a significant factor.

Furthermore, the column-based protocols are not suited to high-sample numbers because common bench-top centrifugesare limited to 24—30 tubes per run, whereas commercial high-throughput nucleic acid isolation kits are overly expensive and therefore not appropriate for smaller laboratories with limited budgets. We have developed a high-throughput plasmid DNA isolation protocol using silica-coated magnetic beads.

For this, bacterial colonies are grown in a 2. The plasmid DNA is then captured and immobilised with silica beads, and remaining particles cell debris, proteins, etc. Whereas BOMB protocol 5. Upon addition of guanidinium-containing binding buffer, plasmid DNA can be immobilised on fresh magnetic beads and further purified. Amount, purity, and quality of the extracted plasmid DNA are comparable to commercial preparations, and the isolated DNA is suitable for both restriction digestion and Sanger sequencing S3B and S3C Fig or other common downstream applications.

Using silica-coated BOMB beads, we routinely process up to samples in 3 to 4 hours for approximately 0. Complete costs per 96 samples were calculated, taking into account plastics, solvents, and enzymes like DNase I. These costs were omitted for the kit content replacement cost column. The isolation of TNA, genomic DNA, or total RNA from bacteria and eukaryotic cells is a basic wet-lab technique and is the starting point for many molecular biology experiments. A classical method for DNA involves lysis of cells in a low-salt buffer with Proteinase K and a detergent, followed by phenol-chloroform extraction and ethanol precipitation [ 24 ].

For RNA, various protocols have been developed over the years [ 25 — 27 ]; however, the most common involve guanidinium species as a strong denaturant that suppresses RNase activity [ 28 ] and facilitates binding of RNA to the silica bead. Therefore, experiments with a large number of samples are impossible for most laboratories. We have found that for bacterial and mammalian cells without extensive extracellular protein, TNA can be efficiently purified using only a sarkosyl and guanidinium-isothiocyanate GITC -based buffer for protein denaturation and cellular lysis Fig 2E—2H , S1 Appendix , BOMB protocols 6.

Following lysis, isopropanol is used to drive precipitation of the nucleic acid to the magnetic beads—BOMB silica beads work well for capture, as do carboxyl beads. The total volume is flexible; however, the relative amount of each component should always be , i. Enzymatic DNase or RNase treatment can be performed either before initial bead purification e. Cells derived directly from solid tissues and organs such as muscle and heart are not easily lysed in GITC without mechanical disruption. In this case, high-throughput lysis and protein digestion can be first performed in a low-salt buffer using Proteinase K, followed by further denaturation in a more concentrated 1.

Although RNA is not well preserved using this high-throughput method, we have found that DNA purification works efficiently on a range of mammalian tissues with either silica or carboxyl beads compared to classical phenol-chloroform extraction Fig 2L , S4 Fig , S1 Appendix , BOMB protocol 6. Further modifications to the initial lysis steps can be added in order to purify nucleic acid from a very broad range of sources.

Methylation of DNA at the fifth position of cytosines in the context of CG dinucleotides is probably the best studied epigenetic modification and likely plays a central role in defining vertebrate cellular identity [ 29 — 31 ]. Bisulfite sequencing is commonly used to study the distribution of 5-methylcytosine in genomes at single base resolution, and it is considered the gold standard in the field [ 32 ].

This method is widely used and can be employed to sequence a wide variety of samples, ranging from single amplicons to whole genomes.

Nucleic acids

It was developed in the s by Frommer and colleagues [ 33 ] and uses sodium bisulfite to sulfonate unmodified cytosines at the sixth position, followed by hydrolytic deamination and desulfonation in alkaline conditions, thereby converting unmodified cytosines to uracils. This reaction is far slower for methylated or hydroxymethylated cytosines at position 5, resulting in selective base conversion, which can be detected by sequencing, allowing site-specific analysis of the methylation status by comparison to the original sequence S6A Fig. One major drawback of this procedure is that bisulfite treatment causes DNA degradation, especially at high incubation temperatures.

Therefore, over the years, the original protocol has been improved to accelerate the conversion procedure [ 34 , 35 ]. The whole procedure takes around 3 to 4 hours for 96 samples, with a hands-on time of less than 1.

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Perhaps the most apparent benefit of the BOMB system is economic. Commercial column-based nucleic acid extraction kits are commonly used in laboratories. These costs have been calculated using high-yield extractions based upon deep-well plates 1—2 ml ; however, further significant cost savings can be made by scaling reaction volumes down to 0. Being at least 10 to 20 times cheaper than commercial column-based protocols, BOMB methods are suitable for large-scale experiments on a budget.

Generally, the most expensive aspects of the BOMB platform are enzymes, like DNase I or RNase A; however, these costs can be greatly reduced if purification of a single nucleic acid species is not required. Other significant costs associated with the BOMB platform include washing solvents such as ethanol and disposable tips and plates; however, these are usually not included in commercial kits and have to be supplied by the user.

Most of the necessary chemicals required for creating the BOMB platform are readily available in standard molecular biology laboratories, and the rest can be purchased from a range of vendors. For the sake of rigour, our protocols list the suppliers we have used; however, we expect that good quality chemicals from an alternative source will show similar performance.

We have developed BOMB protocols as a consequence of an immediate need to process hundreds of samples on a tight budget and with limited workforce. By using magnets to immobilise nucleic acid captured on magnetic beads, centrifugation steps are eliminated. Because of this, these methods are highly scalable and allow easy processing of multiple batches of 96 samples in parallel by a single researcher with a multichannel pipette.

Because of the well format and the lack of dependence on a centrifuge, these protocols are also automation friendly and can be adapted to liquid handling robot systems if available. Compared to kits with single-spin columns, our protocols are much faster when processing many samples simultaneously. However, even when processing only 1 to 10 samples, most of our protocols are at least as fast as commercial alternatives while retaining the cost advantage, high yield, and quality of the isolated nucleic acids. We have found that the time invested in switching to bead-based protocols is very quickly returned, and changing over to a primarily bead-based laboratory actually simplifies and accelerates many experimental processes.

For example, a researcher may wish to undertake genome-wide methylation and expression analysis from the same cultured cell source. Here, a single TNA extraction can be performed—half of the material can be used directly for bisulfite conversion, whereas the rest can be DNase-treated and further purified to obtain pure RNA, which can then be processed into RNA-sequencing libraries.

Converting to a low-cost bead-based system represents an opportunity to transform the research culture of any laboratory in a profound way. By having the capacity to do high-throughput experiments without extra cost, researchers may gain the ability to study entire populations instead of merely sampling, include extra control samples they otherwise would have to forego, or consider analysing separate cell populations rather than relying on averages generated from bulk tissue.

Integration of next-generation sequencing with the BOMB platform provides additional synergy in these respects. Double-ended indexing allows pooling of potentially thousands of individual samples and amplicons in a single run, yet most laboratories are unable to capitalise on this empowering aspect because, until now, they were not equipped to process samples of this number.

The economic benefits of using the BOMB platform are not only relevant for professional scientists—educational institutions such as schools, universities, and community laboratories will also save precious resources by adopting cheap nucleic acid extraction protocols in their curricula. The reliable and easy to follow step-by-step protocols in combination with the lack of highly toxic chemicals or use of expensive equipment make the BOMB platform an ideal tool to introduce students to modern molecular biology techniques.

Each step of these modular pipelines magnifies the benefits of the BOMB platform; we recently purified human cell line RNA, constructed cDNA, and then amplified and cloned over human genes within 3 weeks using BOMB protocols Oberacker and colleagues, in preparation.

Because multiple rounds of plasmid DNA isolation, screening for positive clones, and subcloning were performed, the additive advantage of using BOMB protocols in a well format over conventional targeted cloning approaches was immense. Initially, all that is required for a switch to the BOMB platform are beads and a magnetic rack, so getting started is easy. And although we are confident all laboratories can make their own beads, one barrier to starting with the BOMB platform can be removed by buying commercially available silica- or carboxyl-functionalised beads. Furthermore, the community can use this platform to expand the repertoire of procedures and sample types in a collaborative manner by discussing existing protocols as well as providing adaptations and completely new protocols.

Here, we provide a set of simple, step-by-step protocols for extraction, purification, and manipulation of nucleic acids from various sources. These protocols can serve as a starting platform for further development of other functionalised MNPs as well as protocols tailored to the specific experimental needs of the users. Currently, our focus is on nucleic acids; however, we expect that further bead-based protocols will continue to be developed for more diverse applications. For example, both the carboxyl and silica coatings can be further chemically derivatised by attaching additional functional groups, like cofactors, proteins, or antibodies e.

Our community-focussed website and forum will facilitate this development and allow troubleshooting, reagent sharing, and the distribution of new user-developed protocols. We envision that better access to magnetic bead technology will drive greater efficiency of research in the life sciences and further empower our collective quest for knowledge. Underlying data for S2 Fig can be found in S2 Data. Margaret I. King Library South. Special Collections Research Center. King Library North. All branches have places for individuals to study, but these branches have additional resources.

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