The Chemists Companion Guide to Patent Law: A Case-Based Approach
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Polar compounds such as DNA have a high tendency of attachment to polar stationary phase under specific ionic strength [ 46 ]. First described by Boom et al. NA binds to the silica present in DE, following the same principles of binding to silica matrices. This procedure has the advantage of reduced pipetting error, shorter protocol time, and less number of steps for sample preparation, being used for plasmid as well as for single or double-stranded nucleic acids [ 52 ]. However, this technique is not routinely used because of comparably high cost. Magnetic beads technology is one of the emerging strategies for extracting RNA and genomic, plasmid, and mitochondrial DNA.
The technique involves the separation of nucleic acids from complex mixtures via complementary hybridization [ 53 ]. In recent years, functionalized magnetic particle or beads have been coupled to suitable buffers systems for a rapid and efficient extraction procedure [ 54 ]. The lack of centrifugation steps that can produce shear forces and cause breaking of nucleic acids is thought to better maintain intact longer fragments from genomic DNA. Usually, it is enough to apply a magnet to the side of a vessel or tube containing the sample mixed with the functionalized magnetic beads and exclusively aggregate the target particles near the vessel wall.
The positive aspect of this technique is avoiding centrifugation steps as well as providing an alternative way for automation of extraction procedures from a large number of samples. The extraction technique can be used in batch processes with a multitude of samples blood, tissues, and others and is relatively easy to execute, being one of the best choices for automation, high-throughput applications, and high sample processivity [ 55 , 56 ]. This method is also suitable for using in low technological environments because it is virtually equipment-free.
Unlike silicate negative charge, anion exchange resin makes use of the positively charged diethylaminoethyl cellulose DEAE to attract the negatively charged phosphate of nucleic acid. So, pH and salt concentration are the important aspects determining the binding or elution of NA to the anion exchange resin [ 58 ].
Anion exchange has the advantage of extracting very pure DNA as compared to silica and the ability to reuse the resin upon renaturation. However, this method used high-salt concentration in the elution step, thus requiring desalting for downstream applications. Absorbent cellulose-based paper is an interesting matrix for nucleic acids purification and storage. Cellulose is a polymer of glucose and therefore highly hydroxylated, producing a polar attraction strong enough to bind nuclei acids under specific chemical conditions. Once cells are spotted onto the paper, the detergent lyses the membranes and EDTA chelates metal ions that are cofactors to nucleases, also preventing the growth of contaminating organisms [ 59 , 60 ].
Hence, when dried, nucleic acids are relatively well protected from the environment, especially due to the unavailability of water molecules.
In fact, DNA extraction from dried blood spotting has been successfully used for PCR-mediated human diagnostics for more than 20 years [ 61 — 63 ]. Although FTA cards have many advantages regarding the easiness of use and storage, processing them to extract good yields of nucleic acids might be more complicated than expected, especially in diluted samples [ 64 ]. Table 3 summarizes the main advantages and disadvantages of most commonly used solid-phase extraction methods. Table 4 gives examples of commercially available kits using the methods described herein, as well as giving typical yields for NA extraction.
Examples of commercially available kits applying each extraction method and typical yields for distinct samples. The binding element in spin-column systems is usually composed of glass particles or powder, silica matrices, diatomaceous earth, and ion exchange carriers. Nucleic acid binding is thus optimized with specific buffer solutions and extremely precise pH and salt concentrations [ 4 ].
Column-based NAE is one of the best techniques among the options available, playing a vital role in ion exchange methods, as it provides a robust stationary phase for a rapid and reliable buffer exchange and thus NAE. This method is fast and reproducible, and its main drawback is the need for a small centrifuge as equipment requirement. Magnetic particle or beads are the first option to eliminate centrifuge-dependent steps in the extraction process. Magnetic beads make use of different ligands such as antibodies, antigens, oligonucleotides, or aptamers, which bind specifically to its target in sample.
The first magnetic particle used for extraction consisted of an iron-oxide core covered by functional carboxylic group, which then binds DNA or RNA [ 81 ]. Since then, many modifications have been made using different surface functional group, such as sulphate, amino, and hydroxyl groups [ 82 ].
Besides these functional groups, preactivated magnetic beads with different functional groups are available such as tosyl or epoxy groups [ 78 , 83 ]. Magnetic beads activated with protein A, protein G, or streptavidin are commercially available. Magnetic bead separation presents many benefits over centrifuge-dependent extraction process by allowing an equipment-free process. Equipment-free separation of NA is also possible with nonmagnetic beads, where the beads are trapped inside plastic bubble pipets [ 84 ]. The increase in growth of diagnostic tests and patient numbers highlights the need for automation in life sciences [ 85 ].
To fulfill this demand, various automated devices have been developed and introduced in the market. The most successful examples are the automated liquid handling robots, which are routinely used in many life science and clinical analysis laboratories for dispensing precise amount of sample, reagents, or other liquids to designated containers. Because of this technology, it is now possible to handle many samples simultaneously with precision and rapidity.
In addition, barcode readers are an integral part of such equipment, allowing for easy traceability of samples and results. Fully automated NAE protocols have been developed for such equipment, using either solid-phase or magnetic beads methods [ 79 ]. However, high sample processivity is a positive aspect of automation while maintaining the sensitivity can be compromised, as low-copy NA targets might be lost [ 86 ]. Small versions of these robots are available and could be useful in laboratory settings with minimal infrastructure. Liquid handling robots certainly have a niche in life sciences and clinical laboratories, but not as POC devices.
Nucleic acid-based detection NAT is preferable compared to immunoassay-based detection because of sensitivity and specificity, but NAT-based diagnosis requires complex infrastructure, sophisticated machinery, and trained personnel. To overcome this hurdle, microfluidic chips have been designed and produced, carrying, on inner chambers, all necessary reagents for molecular based tests as a part of POC-Dx strategy. The union of automation with the need for miniaturization in POC devices led to the development of cartridges that perform one or several biological reactions in a closed container.
These reactions comprise most of the current molecular biology methods, such as NAE, amplification, and identification, as well as serological signatures analyses. Microtubes, pumps, and rotary drives transfer liquids into the specific cartridge chambers where washing, purification, and concentration of nucleic acids take place.
The next step is the movement of the extracted NA to a reaction chamber where real-time PCR happens [ 88 ]. A recent systematic meta-analysis study reviewed hundreds of papers concluded that GeneXpert was the most cost-effective strategy for POC-Dx of Mtb, although its performance was evaluated solely in clinics and primary care centers [ 89 ].
However, it is undisputed that GeneXpert is a breakthrough in NA testing. The FilmArray 2. Specific pouches are used to amplify different targets present in the sample, using Nested PCR, followed by real-time PCR with chemistry-based detection. The software then automatically generates identification reports using DNA melting analysis based on specific control reactions or a melting curve database of known sequences. Table 5 presents a summary of the devices available most commonly used in NAE protocols.
A major obstruction for the development of a complete and easy-to-use solution for POC-Dx is the integration of sample preparation protocols into the portable devices. Removing interferents and extracting the target molecules are no trivial task especially due to the vast differences among sample matrices as well as characteristics of the target analytes. While NAE protocols are well established in the laboratory and many advances have been made since the inception of microfluidic Dx devices, commercial availability of these devices is still rare [ 90 ].
Excellent reviews are available discussing the technical difficulties as well as the obstacles for implementation and acceptance of new tests based on new technologies [ 90 — 94 ]. One of properties paramount to the performance characteristics of the plastic materials is chemical inertness, that is, the material to which the active substance of interest will be in contact with will not interact and generate undesirable products, generally classified as extractable or leachable [ 95 ].
Toxicological or functional studies often replace extraction and interaction studies, which would be necessary to determine the levels of extractable or leachable products under a given environmental condition. Studies of structural properties of glassy polymers such as the commonly used thermoplastics polycarbonate PC and polymethylmethacrylate PMMA correlate the polymer solubility when exposed to several solvents to the extent of stress cracking [ 96 ].
An advantage of PMMA is its high optical transparency into the ultraviolet range, while PC offers a compatibility with a wider range of solvents and a higher glass transition temperature well suited to applications such as polymerase chain reaction for NA amplification [ 97 ].
However, neither of these is good enough to be used with the chemicals routinely used for NAE. For example, PMMA cannot be cleaned by strong solvents such as acetone or methanol, because these chemicals would significantly damage its surface and decrease transparency [ 97 ] Table 6. Chemical compatibility of various chemicals used in nucleic acid extraction procedures and plastic polymers commonly used in microfabrication. These changes can happen due to several physicochemical reactions, such as i chemical interaction with polymer chain which can disturb their structure and result in depolymerization; ii physical interaction, that is, adsorption of chemicals into the plastics, which results in swelling and softening; or iii stress-associated cracking may happen due to the stress-cracking agents, such as plasticizers, or adhesives used during the manufacturing of polymer parts, or even detergents or oils used during the molecular biology processes [ 98 ].
Table 6 lists the effect of the chemicals most commonly used in NAE on the plastics most commonly used for microfabrication of microdevices. Alterations induced by any chemical, as minor as it seems, need to be thoroughly evaluated. Finally yet importantly, there is concern about the volume of sample needed to obtain a meaningful results [ ]. Because the volume of buffers and, therefore, of harsh chemicals used for cell lysis is directly proportional to the volume of the sample, POC-Dx tests are most useful in illness where the pathogen is present in higher counts, such as virus and most bacterial infections.
Parasitic infections, however, present a challenge to POC-Dx because parasite loads can get very close to the limits of detection of the techniques used [ ], thus greatly affecting the availability of target NA in the sample. The volume of the reagents is also important to assure proper mixing of solutions without the common laboratory instruments because small volumes are easier to homogenize [ ]. Lessons learned from previous attempts in developing diagnostic tests have taught us that availability of the best possible POC-Dx test is not enough.
Its implementation is also very important and often underestimated, since only few diseases have a validated POC-Dx, such as HIV or malaria [ , ]. Implementation should be considered during the development phase of the POC-Dx, so that end-users are identified, their level of experience is assessed, and the developing test is used at the right lab tier [ 92 ].
The main idea is to provide low cost and timely effective healthcare to the patient and quick decision making for healthcare providers. Paper-based devices are abundant, either directly operating or directing the biochemical, serological, or nucleic acid reactions [ , ]. Because they are easily manipulated to attach recognition molecules antibodies, enzymes, proteins, nucleic acids, etc.
Unlike protein or metabolite-based POC tests, one of the major challenges for nucleic acid-based POC tests is the need to consolidate three distinct protocol procedures into a single device: 1 nucleic acid extraction; 2 amplification; and 3 detection. Development of a nucleic-based testing device that is specific, sensitive, portable, and relatively easy to operate has presented several challenges that have been elegantly reviewed elsewhere [ 90 ]. Development of an ideal NAE method for POC is impaired by many factors and researchers are still in quest for a suitable solution.
However, neither method is yet good enough for widespread implementation in POC-Dx methods. Solid-phase extraction depends on centrifugation, while magnetic beads require an external magnet source for mixing. In this aspect, magnetic beads are favored because implementation of magnetic stirring in POC-Dx devices is somewhat easier than implementation of separation through stationary membranes. Although both rely on the use of chaotropic reagents for lysing cells and releasing the NA from the scaffold and structural nucleic proteins, washing steps are more efficient in beads-based methods.
The main challenges in implementing molecular biology-based systems in resource-constrained areas are the high cost of instrument and reagents, as well as lack of reliable infrastructure and continuous maintenance support and temperature maintenance devices [ 88 ]. Proper disposal of biological waste generated by medical tests is also a concern, not to mention that some waste is chemical and requires special treatment before disposal e. After almost years after the first successful isolation of DNA by Friedrich Miescher, nucleic acids are now central to obtaining biological information in areas as distinct as specimens' identification for conservational purposes to the realms of personalized medicine and pharmacogenomics.
Protocols and devices used for NAE have evolved from thiocyanate-phenol-chloroform manual techniques to user-friendly column-technology and automated platforms, but no general gold-standard method has yet been established. This review analyzed the working principle of each available method, as well as their advantages and disadvantages.
The take-home message is that each application has specific characteristics, which should then guide each researcher to the most suitable method. Although molecular biology techniques are sensitive and accurate methods, they require a rather well established laboratory setting and expensive instruments, as well as skilled personnel to run the tests and analyze the results, which are not always available.
In the last years, lab-on-chip technology has brought the promise of taking the management of biological information where it is needed, such as low-resource settings, a doctor's clinic or a hospital patient bedside. However, although progress has been made, several obstacles still hamper the use of NAE protocols in POC-Dx tests, as it can be seen by the low number of products using lab-on-chip technology.
Overcoming the challenges and limitations of NAE protocols will greatly increase the use of molecular biology techniques and thus increase the overall quality of life of the general population by providing access to better diagnostic tests. The authors are grateful to Dr. Marchini and Dr.
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Adriana C. Umaki for critical reading of the manuscript. The authors declare that there are no conflicts of interest regarding the publication of this article. National Center for Biotechnology Information , U. Journal List Biomed Res Int v. Biomed Res Int. Published online Jul Author information Article notes Copyright and License information Disclaimer. Received Mar 31; Accepted Jun 5. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This article has been cited by other articles in PMC. Abstract Nucleic acid extraction NAE plays a vital role in molecular biology as the primary step for many downstream applications. Introduction Nucleic acid extraction NAE is one of the most pivotal steps in molecular biology, being routinely used in many areas of the biological and medical sciences, as this procedure marks a starting point in any molecular diagnostic kit [ 1 ]. Table 1 Main characteristics of chemical and mechanical methods to extract nucleic acid adapted from Harrison Open in a separate window.
Chemically Driven Methods These methods rely on biochemical properties of the cellular components to elicit the desired molecular separation and might exhibit preference or exclusivity in extracting DNA or RNA, depending on its intrinsic characteristics. Cetyltrimethylammonium Bromide CTAB Extraction Cetyltrimethylammonium bromide extraction method is mainly used for plant samples and their parts, such as leaves, seeds, and grains.
Table 2 Summary of advantages and disadvantages of the main NAE methods. Silica Matrices In , it was found that silicates have high binding affinity for DNA under alkaline conditions and increased salt concentration [ 44 ]. Glass Particles Glass particles, whether in powder as chromatography stationary phase or in microbeads form, have also been used for extraction of nucleic acids. Cellulose Matrix Absorbent cellulose-based paper is an interesting matrix for nucleic acids purification and storage. Table 3 Summary of the advantages and disadvantages of solid-phase extraction methods.
Table 4 Examples of commercially available kits applying each extraction method and typical yields for distinct samples. Devices Used in Extraction Methods 4. Spin Columns The binding element in spin-column systems is usually composed of glass particles or powder, silica matrices, diatomaceous earth, and ion exchange carriers. Beads or Magnetic Beads Magnetic particle or beads are the first option to eliminate centrifuge-dependent steps in the extraction process. Automation Liquid Handling Robots The increase in growth of diagnostic tests and patient numbers highlights the need for automation in life sciences [ 85 ].
Table 5 Summary of available devices used in nucleic acid extraction protocols. Limitations for Implementation of Extraction Protocols in Portable Devices A major obstruction for the development of a complete and easy-to-use solution for POC-Dx is the integration of sample preparation protocols into the portable devices.
Table 6 Chemical compatibility of various chemicals used in nucleic acid extraction procedures and plastic polymers commonly used in microfabrication. Challenges for Implementation in POC Diagnostic Tests Lessons learned from previous attempts in developing diagnostic tests have taught us that availability of the best possible POC-Dx test is not enough. Conclusion After almost years after the first successful isolation of DNA by Friedrich Miescher, nucleic acids are now central to obtaining biological information in areas as distinct as specimens' identification for conservational purposes to the realms of personalized medicine and pharmacogenomics.
Acknowledgments The authors are grateful to Dr. Conflicts of Interest The authors declare that there are no conflicts of interest regarding the publication of this article. References 1. Tan S. Journal of Biomedicine and Biotechnology. Doyle K. Lesk A.
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Analytical Chemistry. Merkle S. Recent developments and applications of solid phase microextraction SPME in food and environmental analysis—a review. Nielsen A. Trace determination of volatile sulfur compounds by solid-phase microextraction and GC-MS. Vogelstein B. Preparative and analytical purification of DNA from agarose. Proceedings of the National Academy of Sciences. Esser K. Nature Methods. V Padhye V. United States patent US Promega Corporation; Nucleic acid purification on silica gel and glass mixture. Mutin A. Through abundant examples from case law as well as real-world situations with which a researcher might be faced, this book provides readers with a better understanding of how to put that knowledge into practice.
Unfortunately, most researchers involved in testing and evaluating novel chemicals have a limited knowledge of laws that pertain directly to their work. This thorough, accessible reference fills this critical educational gap by delivering relevant legal concepts in a clear, concise manner free of specialized jargon. This indispensible reference:.
Miller has over a decade of research experience as a medicinal chemist in the pharmaceutical industry and has worked as an associate at a law firm as well as served as in-house patent counsel in big pharma and the biotech industry. Miller is licensed to practice law before the state bar of Pennsylvania and the United States Patent and Trademark Office.
He currently is practicing both medicinal chemistry and patent law in the Boston area. MARK J. EVANS obtained his PhD from Northwestern University and his MBA from Penn State University; has fifteen years of pharmaceutical industry experience in the development of both small molecule and protein therapeutics at Wyeth Pharmaceuticals and Alexion Pharmaceuticals; and currently works in regulatory affairs in the Philadelphia area.
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