DNA Extractions -the Past, Present and Future Approaches

DNA Extractions - the Past, Present and Future Approaches Nur Munirah Bt Majid, Uda Hashim*, Subash C.B. Gopinath, and xxxxxxxxx Biomedical Nano Diagnostics Research Group, Institute of Nano Electronic Engineering (INEE), Universiti Malaysia Perlis (UniMAP), 01000 Kangar, Perlis, Malaysia. Correspondence to: Prof. Uda Hashim Biomedical Nano Diagnostics Research Group, Institute of Nano Electronic Engineering (INEE), University Malaysia Perlis (UniMAP), 01000 Kangar, Perlis, Malaysia. Email: [email protected] Abstract DNA, RNA and protein are the major macromolecules play important roles in the functional aspects of every living thing. As stated by ‘central dogma’ DNA is the primary molecules not only as building block of human genetics but also towards medical diagnosis and forensic applications, due to high specificity of DNA and they varies from one individual to another. DNA molecules in native condition are in the complex form with other macromolecules in most of the cases to fulfill functions. This complex formation makes the necessity of separation (extraction) of DNA molecules from others before being applied for sensing or forensic applications. Moreover, as technologies develop, there is an urge toward method to extract macromolecules that specific to particular species. The processes of extraction and purification of DNA used previously were complicated, time-consuming, labor-intensive, and limited in terms of overall throughput, but now-a-days there are many specialized and sophisticated methods that can be used to extract DNA in pure form. Current technologies should allow a high-throughput of samples to be extracted for DNA, however the crucial developmental process of DNA extraction during transitional stages of developments were not explored properly. In this overview, we gleaned the extraction methods demonstrated from past to present and for a future with their critics. Key words: DNA, Extraction, Purification, Macromolecule, Nucleases 1. Introduction Nucleic acid extraction and purification is one of the essential steps should follow the workflow of genetic analyses and considered to be a most crucial method used in molecular biology. Biological macromolecules including deoxyribonucleic acid (DNA), can be isolated from biological materials such as living or conserved tissues, cells, virus particles, plant material or other samples. Thus, extractions of macromolecules becoming the basic method that are commonly used in molecular studies. Further, DNAs should be isolated from other complexed macromolecules, as DNAs are carrying basic genetic information for subsequent downstream processes and analytical or preparative purposes. DNA purification involves two major categories that are isolation of recombinant DNA such as plasmids or bacteriophage and the isolation of chromosomal or genomic DNA from prokaryotic or eukaryotic organisms (Doyle, 1996). A successful use of available downstream applications will gain with high-quantity and quality DNA output (Pyzowski & Tan, 2007). There are three important steps that will lead to successful nucleic acid extraction, firstly the effective disruption of cells or tissue, secondly denaturation of nucleoprotein complexes using appropriate reagents to inactivate the nucleases, for example, RNase for RNA removal and DNase for DNA removal and lastly away from contamination (Wink, 2006). The target nucleic acid should be free of contaminants including protein, carbohydrate, lipids, or other factors that will lead to impure output. So, it is essential to choose a suitable extraction method, and thus, few considerations have to be made when evaluating the available options. These may include technical requirements, time efficiency, cost-effectiveness, as well as biological specimens to be used and their collection and storage requirements. Extraction methods involve in DNA isolation becoming more effective and efficient as extraction kits being developed which containing most of the components needed to isolate nucleic acid, but still additional steps are needed to remove other components depending on the type of specimen, which are time consuming and tedious. To overcome these problems, automated systems being designed for medium-to-large laboratory scales have grown in demand over recent years. The prime issues with DNA extraction is to increase the yield and purity of desired product, reproducibility, and scalability of the molecules as well as the speed, accuracy, and reliability of the assay, while minimizing the risk of cross-contamination. Overall, to generate an efficient DNA extraction method, even though proposed methods are seems to be simple, there are critics and rationales with the methods in respect to the purpose. To pin-point and understand more about the crucial steps involved in different methods will make the way to generate a finely tuned method, will be a common strategy suitable to the laboratories and industries. In this overview, the important steps involved in the DNA extraction and purification methods formulated in the past, present and a direction towards future developments are narrated. This study is necessary due to the active participation of DNA molecules in several instances in wide range of studies. DNA is a long polymer of repeating sub-unit called nucleotides, and each nucleotide consists of three sub-units of pentose or five-carbon sugar, a phosphate group and an organic nitrogenous (nitrogen-containing) group (Cseke et al, 2004). DNA is a complex molecule that contains all of the information necessary to build and maintain an organism. In fact, nearly every cell in a multicellular organism possesses the full set of DNA required for that organism. However, DNA does more than specify the structure and function of living things (Mitnik et al., 2012). Whenever organisms reproduce, a portion of their DNA is passed along to their offspring, this transmission of all or part of an organism's DNA helps to ensure a certain level of continuity from one generation to the next, while still allowing for slight changes that contribute to the diversity of life (Buckingham & Flaw, 2007). DNA encodes the genetic information used to assemble proteins in similar to the way the letters on books encode information (Blin & Stafford, 1976). Unique among macromolecules, nucleic acid are able to serve as templates to produce precisely copies of them. 2. Discovery of DNA as main building block Even though, initially the first nucleic acid discovery was realized by a few people, nucleic acid was first identified in the year 1869 by the Swiss physiological chemist, Friedrich Miescher that was called as "nuclein" resides in the nuclei of human white blood cells. The term "nuclein" was later changed to "nucleic acid" and eventually to "deoxyribonucleic acid," or "DNA (Whitford, 2005). At first, Miescher's plan was to isolate and characterize the protein components of leukocytes or white blood cells. For that, he made arrangements for local surgical clinics to send him the used, pus-coated patient bandages for his research. Once he received the bandages, he planned to wash them, filter out the leukocytes, extract and identify the various proteins within the white blood cells until he came across a substance from the cell nuclei that had chemical properties unlike any proteins, including a much higher phosphorous content and resistance to proteolysis, Miescher realized that he had discovered a new substance (Dahm, 2008). Other scientists, Phoebus Levene from Russian biochemist have continued to investigate the chemical nature of the molecule formerly known as ‘nuclein’ and publishing more than 700 papers on the chemistry of biological molecules over the course of their career (Haines et al, 2005). Levene was the first person to discover the order of three major components (phosphate-sugar-base) of a single nucleotide and the first discovered the carbohydrate component of DNA (Brent, 1998). Levene also proposed that nucleic acids were composed of a series of nucleotides, and that each nucleotide was in turn composed of just one of four nitrogen-containing bases, a sugar molecule, and a phosphate group. Levene made his initial proposal in 1919, discrediting other suggestions that had been put forth about the structure of nucleic acids (Kojima & Ozawa, 2002). Erwin Chargaff, an Australian biochemist was one of a handful of scientists who expanded Levene's work by uncovering additional details of the structure of DNA, thus further paving the way for Watson and Crick model, which demonstrated that hereditary units, or genes, are composed of DNA. As his first step in this search, Chargaff set out to see whether there were any differences in DNA among different species (Dahm, 2004). After developing a new paper chromatography method for separating and identifying small amounts of organic material, Chargaff reached two major conclusions. First, he noted that the nucleotide composition of DNA varies among species. In other words, the same nucleotides do not repeat in the same order, as proposed by Levene. Second, Chargaff concluded that almost all DNA, no matter what organism or tissue type it comes from, maintains certain properties, even its composition varies (Kojima & Ozawa, 2002). In particular, the amount of adenine (A) is usually similar to the amount of thymine (T), and the amount of guanine (G) usually approximates the amount of cytosine (C). This second major conclusion is now known as "Chargaff's rule." Chargaff's research was vital to the later work of Watson and Crick model, but Chargaff himself could not imagine the explanation of these relationships and specifically, that A bound to T and C bound to G within the molecular structure of DNA (Brooks, 1998). Chargaff's realization that A = T and C = G, combined with some crucially important X-ray crystallography work by English researchers Rosalind Franklin and Maurice Wilkins, contributed to Watson and Crick's derivation of three-dimensional, double-helical model for the structure of DNA. Watson and Crick's discovery was also made possible by recent advances in model building, or the assembly of possible three-dimensional structures based on known molecular distances and bond angles. (Sambrook & Russel, 2001). 3. DNA extraction discovery As stated above, Friedrich Miescher, the first scientist attempted to isolate DNA while studying the chemical composition of cells. Firstly, he was aimed in solving the fundamental issue or principles in life by determine the composition of the cell. In 1869, he tried to isolate cells in lymph nodes but the purity of lymphocyte was impossible to obtain, thus he switch to use leukocytes that he has collected from the samples on fresh surgical bandages and conducted experiments to purify and classify proteins in these cells. He was mainly concentrating in various proteins that make leukocyte, but during his experiments he accidently identified a novel substance in the nuclei, and he called “nuclein” (Dahm, 2004). He has developed two protocols to separate cell’s nuclei from cytoplasm and to isolate this novel compound, a so-called DNA, which is differed from proteins and other cellular substances. He noticed that a substance precipitated from the solution, when acid was added and dissolved, when alkali was added. Thus, for the first time he had obtained a crude precipitate of DNA (Brooks, 2002). However, his first protocol was believed to be failed to yield enough material to continue with further analysis. Then, he had developed a second protocol to obtain larger quantities of purified nuclein, which had been named as ‘nucleic acid’ later by his student, Richard Altman. This scientific finding, together with the isolation protocols being standardize and was published in 1871 in collaboration with his mentor, Felix Hoppe-Seyler. However, in the year 1958, Meselson and Stahl have developed a routine laboratory procedure for DNA extraction. They performed DNA extraction from bacterial samples of Escherichia coli using a salt density gradient centrifugation, resulting in DNA extraction techniques that can perform on various types of biological sources (Melkonyan et al., 2008). These developments formed the basis for DNA extraction methods developed in the later stages, which follow the important facts that mentioned earlier, as they need effective disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and other enzymes, removal of biological and chemical contaminants, and finally obtained the pure DNA as precipitants. Most of the conventional methods or other modified methods follow similar basic steps and included the use of organic and non-organic reagents and centrifugation. These basic steps finally entered into varieties of automated procedures and commercially available kits. 4. Conventional extraction methods After the Miescher achievement to obtain DNA from cell, many scientists interested and followed the lead, eventually to further advancements in the DNA isolation and purification protocol were attained. The first laboratory procedures developed for DNA extraction were from density gradient centrifugation strategies. This was proposed by Meselson and Stahl in the year of 1958 to demonstrate semi-conservative replication of DNA (Buckingham & Flaws, 2007). Later protocol was mainly based on the use of differences in solubility of large chromosomal DNA, plasmids, and proteins in alkaline buffer, now-a-days there are many specialized methods of extracting pure DNA. General extraction protocols are divided into solution-based and column-based and most of these protocols have been implemented in commercial kits that ease the DNA extraction processes. Phenol-chloroform extraction Phenol-chloroform is a  liquid-liquid extraction  technique in biochemistry and molecular biology studies to purify nucleic acids and eliminate the proteins. In brief, aqueous samples are mixed with equal volumes of phenol:chloroform mixture. After mixing, the mixture is centrifuged and two distinct phases are formed, because phenol:chloroform mixture is immiscible with water. The aqueous phase is on the top, due to its less density than the organic phase (phenol:chloroform). Proteins will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. The upper aqueous phase is pipetted off and this procedure is often performed multiple times to increase the purity of the DNA (Chomczynski & Sacchi, 2006). By mixing two phases, and allowing the phases to be separated by centrifugation, chloroform and phenol mixture is more efficient, as it denature the proteins than either reagent is alone. The phenol-chloroform combination reduces the partitioning of poly (A)+ mRNA into the organic phase and reduces the formation of insoluble nucleic acid and protein complexes at the interphase (Dederich et al., 2002). Moreover, phenol retains about 10-15% of the aqueous phase, which results in a similar loss of nucleic acid. Chloroform also prevents this retention of water and thus improves the yield (Massart, 1981). Typical mixtures of phenol to chloroform are 1:1 and 5:1 (v/v). At acidic pH, 5:1 ratio results in the absence of DNA from the upper aqueous phase; whereas 1:1 ratio, providing maximal recovery, will maintain some DNA present in the upper aqueous phase (Gjerse et al, 2009). For example, a very polar solute such as urea is soluble in highly polar water, less soluble in fairly polar methanol, and almost insoluble in non-polar solvents, such as chloroform and ether (Esser et al., 2006). Nucleic acids are polar, because of their negatively charged phosphate backbone, and therefore nucleic acids are soluble in the upper aqueous phase instead of the lower organic phase (water is more polar than phenol) (Woodard et al., 1994). As contrast to protein, contain varying proportions of charged and uncharged domains, producing hydrophobic and hydrophilic regions (Buckingham & Flaws, 2007). In the presence of phenol, the hydrophobic cores interact with phenol, causing precipitation of proteins and polymers (including carbohydrates) to collect at the interface between two phases (often as a white flocculent) or for lipids to dissolve in the lower organic phase (Massart, 1981). The pH of phenol determines the partitioning of DNA and RNA between the organic phase and the aqueous phase (Arnold et al, 2005). At neutral or slightly alkaline pH (pH 7-8), the phosphate diesters in nucleic acids are negatively charged, and thus DNA and RNA both partition into the aqueous phase. DNA is removed from the aqueous layer by lowering the pH to 4.8. At this acidic pH, most proteins and small DNA fragments (<10 kb) fractionate into the organic phase and large DNA fragments and some proteins remain at the interphase (Arnold et al, 2005; Whitford, 2005). Acidic phenol retains RNA in the aqueous phase, but moves DNA into the phenol phase, because the phosphate groups on the DNA are more easily neutralized than those in RNA and an acid pH also minimizes RNase activity (Watson et al, 2004). Isoamyl alcohol is sometimes added to prevent foaming (typically in a ratio of 24 parts chloroform to 1 part isoamyl alcohol). Guanidinium salts are also used to reduce the effect of nucleases (Puissant & Houdebine, 1990). Alkaline lysis Alkaline lysis is the method of choice for isolating circular plasmid DNA, from bacterial cells. It was first described by Birnboim and Doly in 1979 and with a few modifications, been the preferred method for plasmid DNA extraction from bacteria. It has been proven to work well with all strains of E. coli and with bacterial cultures ranging in size from small scale (1 mL) to large scale (500 mL) in the presence of Sodium Dodecyl Sulfate (SDS) (Cui et al, 2003). The principle in the alkaline lysis procedure, bacterial cells is exposed to Sodium hydroxide (NaOH) and SDS which are strong detergents. Eventually, this will cause the cell walls and membranes to burst and the contents of the bacteria are spilled out (Feng et al., 2004). An acidic solution of sodium acetate is then added to neutralize the solution. At this point, most of the cell membrane material and the genomic DNA precipitate to form a phlegm-like mass. The cell contents (including plasmids) can be separated from this material by centrifugation. The resultant supernatant is then extracted to purify the plasmid DNA (Tinay et al., 1998; Dai et al., 2001; Cui et al., 2006). It is one of the most general useful techniques as it is a fast, reliable and relatively clean way to obtain DNA from cells. In this method, rapid anneal the following denaturation, allows the plasmid DNA to be separated from the bacterial chromosome. CTAB extraction method This method has been shown to give intact genomic DNA from plant tissue (Simon, 1996). The initial step that needs to extract the plant genomic DNA is to grind the sample by freezing it with liquid nitrogen to break down the cell wall material and allow access to DNA while harmful cellular enzymes and chemical remain inactivated. After grinding the sample, it can be re-suspended in a Cetyltrimethylammonium bromide (CTAB) buffer. CTAB is a non-ionic detergent that can precipitate nucleic acids and acidic polysaccharides from low ionic strength solutions (Sambrook & Russel, 2001). In order to purify DNA, insoluble particulates are removed through centrifugation while soluble proteins and other material are separated through mixing with chloroform and centrifugation. DNA must then be precipitated from the aqueous phase and washed thoroughly to remove contaminating salts. The purified DNA is then re-suspended and stored in TE buffer or sterile distilled water (Schott & Arnold, 1994). This method has been shown to give intact genomic DNA from plant and other tissues. To check the quality of the extracted DNA, a sample is run on an agarose gel, stained with ethidium bromide and visualized under UV light. Ethidium bromide (EtBr)-Cesium Chloride (CsCl) gradient centrifugation The CsCl gradient centrifugation is a complicated, expensive, and time-consuming method compared to other purification protocols and requires large scale bacterial culture to perform. Therefore, this protocol is not suitable for the mini-preparation of plasmid DNA (Cseke et al., 2004). The desired nucleic acids can be concentrated by centrifugation in an EtBr-CsCl gradient after alcohol precipitation and re-suspension. The main principle is the intercalation of EtBr that will alters the density of the molecule in high molar CsCl. Thus, the closed circular molecules will accumulate at lower densities in the CsCl gradient, because they incorporate less EtBr per base pair compared to linear molecules. The hydrophobic EtBr is then removed with appropriate hydrophobic solvents after extraction. The purified nucleic acid then will be re-precipitated with alcohol (Wink, 2006). Solid-phase nucleic acid extraction Solid-phase nucleic acid purification is an alternative toward a conventional method and can be found in most of the commercial extraction kits in the market. This system also promises for quick and efficient purification compared to conventional methods (Esser et al, 2005). There are many drawbacks with liquid-liquid extraction such as incomplete phase separation, repeated centrifugation step and others. Via solid phase system such as silica, glass bead, it can absorb nucleic acid in the extraction process depending on the pH and salt content of the buffer (Guarerro et al., 2010). The absorption process is based on the hydrogen-binding interaction with a hydrophilic matrix under chaotropic conditions. In this system, there are four key steps involved such as cell lysis, nucleic acids adsorption, washing, and elution (Kojima & Ozawa, 2002). The initial step in a solid phase extraction process is to condition the column for sample adsorption. This can be done by using certain type of buffer at a particular pH to convert the surface or functional groups on the solid into a particular chemical form (Gjerse et al., 2009). Next, the sample then being degraded by using lysis buffer and the desired nucleic acid will absorb to the column with the aid of high pH and salt concentration of the binding solution (Smith et al., 2002). Other compounds, such as protein being removed from the desired product by using the washing buffer containing a competitive agent (Padhye et al., 1997). Finally, for the elution step, TE buffer or distilled water is introduced to release the desired nucleic acid from the column by breaking the bond of DNA backbone from the surface of the solid compound, so that it can be collected in a purified state (Smith et al., 2002). Normally, rapid centrifugation, vacuum filtration, or column separation is required during the washing and elution steps of purification process. 7.1. Silica matrices-based nucleic acid purification The basis for most of the products related to nucleic acid purification is the unique properties of silica matrices for selective DNA binding. There are many types of silica materials can be used including glass particles, such as glass powder, silica particles, and glass microfibers prepared by grinding glass fiber filter papers, and including diatomaceous earth (Padhye et al., 1997). Hydrated silica matrix, which was prepared by refluxing silicon dioxide in sodium hydroxide or potassium hydroxide at a molar ratio of about 2:1 to 10:1 for at least about 48 hours, had been introduced in DNA purification (Woodard et al, 1994). The principle of silica matrices purification is based on high affinity of negatively charged DNA backbone towards positively charged silica particles (Esser et al., 2006). Sodium plays a role as a cation bridge that attracts the negatively charged oxygen in the phosphate backbone of nucleic acid (Feng et al., 2004). Sodium cations break the hydrogen bonds between the hydrogen in water and the negatively charged oxygen ions in silica under high salt conditions (pH ≤ 7). In the presence of high salt buffer, DNA will binds to silica particles (Arnold et al., 2005). Then the silica with adsorbed DNA will be washed using washing buffer to remove salt and impurities from the original sample, and the clean DNA will eluted under low ionic strength (pH ≥ 7) either in water or TE buffer (Chen et al., 2007). This isolation method of DNA using silica is said to be faster (approximately 20 min) and easier to perform than the other organic-based extraction method. This method also replaces the Potassium Iodide (KI) based procedure, where free Iodine may modify the purified DNA. When using silica adsorption method for isolating DNA from agarose gels, it is important to note that use of TBE buffer (Tris-borate-EDTA) can inhibit the ability of DNA to bind silica, thus lowering recovery efficiency.  Silica extraction works well with a wide size range of DNA and allows efficient recovery (90%) of product. By using silica, it can purify DNA as small as 100 bp (Salgueiri˜no-Maceira et al, 2006). There is no upper size limit on recovery efficiency of DNA. However, precautions should be taken during purification of longer DNA fragments (20 kb) to avoid shearing (Woodard et al, 1994). Proteins and RNA do not bind to silica and are eliminated during washes and for this reason it is also an ideal tool to purify and concentrate DNA directly from various reaction mixtures. Other advantage is, because silica does not bind to oligonucleotides with high efficiency. This method can also be used to remove low molecular weight oligonucleotides or nucleotides from DNA. Silica can also be used to remove RNA from DNA, because RNA does not bind to silica and will yield only the pure DNA as a final result (Esser et al., 2006). The purified DNA is suitable for any molecular biology procedures such as restriction digestion, cloning, sequencing and etc. Small amounts of silica do not inhibit enzymatic reactions, therefore silica bound DNA can be used directly for PCR or enzymatic cleavages without prior elution of DNA (Woodard et al., 1994). Magnetic bead-based nucleic acid purification Magnetic bead technology is recognized as combining high quality genomic DNA (gDNA) production with compatibility for high-throughput processing. Furthermore, automation of magnetic bead methodology reduces user to be exposed to bio-hazardous human materials. Magnetic separation is known to be a very simple and efficient way which used in purification of any desired nucleic acid. Currently, many magnetic carriers are now commercially available. Particles having a magnetic charge may be removed by using a permanent magnet in the application of a magnetic field. Usually, magnetic carriers often immobilized with affinity ligands or can be prepared from biopolymer tend to show affinity toward the target nucleic acid, are commonly used for the isolation process (Dederich et al., 2002). For example, magnetic particles that are produced from different synthetic polymers, biopolymers, porous glass or magnetic particles based on inorganic magnetic materials, such as surface modified iron oxide are preferred to be used in the binding of nucleic acids, but magnetic particulate materials such as beads are more preferable to be a support in isolation process, because of their larger binding capacity. The nucleic acid binding process may be assisted by the nucleic acid surrounding support and a permanent magnet can be applied to the side of the vessel to aggregate the particles near the wall of the vessel and the sample or desired DNA can be eluted using TE buffer or distilled water (Berensmeier, 2006). Particles having magnetic or paramagnetic properties are employed in an invention where they are encapsulated in a polymer such as magnetizable cellulose (Nargessi, 2005). In the presence of certain concentrations of salt and polyalkylene glycol, magnetizable cellulose can bind to nucleic acids. Small nucleic acid required higher salt concentrations for strong binding to the magnetizable cellulose particles. Therefore, salt concentration can be selectively manipulated to release nucleic acid bound to magnetizable cellulose on the basis of size. The magnetizable cellulose which bound with nucleic acid will be washed with suitable wash buffer before they contact with a suitable elution buffer to separate out the desired nucleic acid with cellulose. Separation of magnetizable cellulose from supernatant during all the purification steps can be done by applying a magnetic field to draw down or draw them to the side of the vessel (Nargessi, 2005). The magnetizable cellulose used in this invention has an iron oxide content of up to around 90% by weight of the total mass of the cellulose. The magnetic component of cellulose can also be substituted by other magnetic compounds such as ferrous oxide or nickel oxide (Berensmeier, 2006). There are many extraction kits that are currently implies this technology, available commercially in the market (Sasso et al., 2012). The special part of this kit is that the reagents provided are intended for use with magnetic tools, such as in GeneCatcher™ extraction kits and other kits that currently available in the market. Magnetic beads are a simple and reliable method of purifying genomic, plasmid and mitochondrial DNA. Under optimized conditions, DNA selectively binds to the surface of magnetic beads, while other contaminants stay in solution (Keeley et al., 2013). Purified DNA can then be used directly (recovered by using elution buffer) for many applications, such as sequencing or restriction digestion. The major advantage of this method is that there is no need for centrifugation or vacuum manifolds, which can be a bottle-neck in many automated processes. Separation can be done manually, semi-automated or fully automated in 24, 96 and 384 well plates. Equipment necessary for this technology includes the bead purification kits, magnetic particle processing systems, magnetic separators, tubes, columns and flasks. Features to be looked are narrow size distribution of the beads and an adequate magnetic mass susceptibility (Sasso et al, 2012). There is also another extraction kit that has the same principle as the extraction described above, which used the magnetic-particle technology for nucleic acid purification that combines the speed and efficiency of silica-based DNA purification with the convenient handling of magnetic particles (QIAGEN Inc., QAsymphony_ DNA Handbook, QIAGEN,Alameda, Calif, USA, 2008). A magnetic rod protected by a rod cover is used for the capture of magnetic particles. It enters a vessel containing the samples and attracts the magnetic particles. Then, the magnetic rod cover is positioned above another vessel and the magnetic particles are released (Applied Biosystems, MagMAXTM Total Nucleic Acid Isolation Kit, Applied Biosystems, Foster City, Calif, USA, 2008). Other than magnetic bead, zirconia bead also can be used in nucleic acid purification. These micro-spherical paramagnetic beads have a large available binding surface and can be dispersed in solution. This characteristic allowed thorough nucleic acid binding, washing, and elution. The extraction kits that use zirconia beads mainly adapt the guanidinium thiocyanate-based solution protocol that not only releases nucleic acid but also inactivate nuclease in the sample matrix (Ma et al., 2013). After the lysis step, dilution of samples is done by isopropanol. Paramagenetic beads are added to the samples for the nucleic acid binding purpose. The mixture of beads and nucleic acid are immobilized on magnets and washed to remove protein and other contaminants. Removal of residual binding solution is done with a second wash solution and finally the nucleic acid is eluted in low-salt buffer (Ma et al., 2013). Glass Particle-based nucleic acid purification Glass particles, powder and beads are useful for nucleic acid purification. The adsorption of nucleic acid on the glass substrate occurs most likely based on the mechanism and principle that similar to adsorption chromatography (Dederich et al, 2002). Nucleic acid purification can also be done on silica gel and glass mixture. This invention has discovered that a mixture of silica gel and glass particles can be used to separate nucleic acid from other substances in the presence of chaotropic salts solution (Padye et al., 1997). Diatomaceous earth Diatomaceous earth, which is also known as kieselguhr or diatomite, has silica content as high as 94% (Little, 1991). It has been useful for the purification of plasmid and other DNA by immobilizing DNA onto its particles in the presence of a chaotropic agent. There are several methods regarding purification of plasmid DNA using this technique has been described in detail. The principle behind this purification technique is almost similar as other purification techniques. The desired DNA will binds to the silica dioxide, which is the major component of the diatomaceous earth. In the presence of chaotrophic salts such as guanidine hydrochloride, NAL or guanidine isothiocyanate, the chaotropic salts will denature the protein and will be washed out using washing buffer. The resin-bound DNA will be collected in plastic column using vaccum manifold and eluted using low salt buffer or in distilled water (Vrancken et al, 1995). Anion-exchange materials Ion exchange chromatography has emerged as a reliable alternative to classic CsCl-ethidium bromide gradients for isolating nucleic acids with the highest purity. The principle is based on the interaction between positively charged diethylaminoethyl cellulose (DEAE) groups on the resin’s surface and negatively charged phosphates of the DNA backbone. Most of the anion-exchange resin consists of defined silica beads with a large pore size and some of the anion-exchange might contain a porous silica that modified with diethylaminoethanol that give a hydrophilic surface coating with a high charge density (Endres et al., 2003). The resin works over a wide range of pH conditions (pH 6–9) and/or salt concentration (0.1–1.6 M) which can optimize the separation of DNA from RNA and other impurities (Knudsen et al, 2001). In this technique, salt concentration and pH conditions of the buffers play the main role that determine whether nucleic acid is bound or eluted out from the column. DNA can bind to the DEAE group over a wide range of salt concentrations. Other unwanted impurities such as protein and RNA usually washed from the resin by using medium-salt buffers, while DNA remains bound until eluted with a high salt buffer (Endres et al., 2003). There are lots of commercially available strong or weak positively charged anion exchanger materials that can be used with selected solutions of known ionic strength for adsorption and elution of desired DNA. The ionic strength for elution is generated by using known salt concentration, which mixed with a buffer to control pH strength, ideally corresponding to the lowest ionic strength at which the nucleic acids will completely elute (Selingson and Shrawder, 1990). Based on the research, plasmid DNA usually isolated well using this technique. Plasmid purification method based on a unique anion exchange membrane (IEXM) was developed for the production of superior quality plasmids (Marion & Warren, 1989). This method was simpler and more efficient as compared to the conventional bead-based methods. Plasmids were extracted from bacterial cells through alkaline lysis and the crude lysate was clarified by a sequential filtration device that not only removed cell debris but micellar aggregates as well (Prazeres et al, 1998). The clarified lysate was mixed with an extraction solution and loaded into a spin column containing IEXM. The binding, washing, and elution conditions were optimized to achieve efficient isolation of plasmids from the impurities. IEXM had an exceedingly highly dynamic binding capacity, excellent selectivity, and a near 100% recovery for plasmids (Prazeres et al., 1998). These products are thought to be cost-effective, disposable, minimize cross contamination and sophisticated chromatographic instrument is not required. All-in-one biomolecules extraction Generally, the extraction or purification techniques or kits available in the market can only allow the extraction of one type of nucleic acid, either DNA or RNA, or protein from a targeted organism. When the sample material is limiting, it is desirable to extract DNA, RNA and protein from the same source. For example, the clinical sample, such as bone tissue of fluids that are painful to collect from the patient should be used wisely. A variation on the single-step isolation method of Chomczynski and Sacchi in 1987 shows that the guanidinium thyicyanate homogenate is extracted with phenol:chloroform at reduced pH, allows the preparation of DNA, RNA and protein from tissue or cells. This method involves the lysis of cells with guanidine isothiocyanate and phenol in a single phase solution and the second phase forms after the addition of chloroform that result in DNA and proteins extracted and leaving RNA in the aqueous supernatant. The DNA and proteins can be isolated from the organic phase by precipitation with ethanol or isopropanol and the RNA precipitated from aqueous phase with isopropanol (Sambrook & Russel, 2001). Currently, as the technologies develop, extraction process also becoming easier, formulated as all-in-one extraction kits have been introduced in the market. For example, a column-based extraction kit that designed to purify genomic DNA, total RNA and total protein from a single biological sample simultaneously, without the usage of toxic substances such as phenol: chloroform and alcohol precipitation and usage of small sample size. The targeted sample does not need to be separated before the purification step (Prazeres et al., 1998). Other than that, solution-based 3-in-1 extraction kit is another example of non-organic solutions kit that can extract and purify DNA, RNA and protein, from different organisms in any types and sizes. The principle is the same as three simple steps protocol, less time consuming (15-30 min) and thus, provides a fast and easier way to do the extraction of different biomolecules and gives higher yield of desired results (DeRiPRO, DNA, RNA and Proteins Extraction Technology). Automated Extraction System When come across automated extraction system, people will think about large, expensive and complex instrumentation designed for high-throughput sample processing. To handle such instrument does need for professional expertise to handle. To overcome the drawbacks from complicated to user friendly instruments, researchers tried to developed many system to simplify the isolation of nucleic acids (Loeffler et al., 2004). Thus, now-a-days with high technology, this system was designed for medium to large laboratories which has grown in presence over recent years (Boyd, 2002). There are many extraction system that is available in the market has met the requirements stated above. Some of the applications, such as forensic department required lot of sample to be process at once. Thus, forensic laboratories need to be fast and reliable sample processing along with high-quality automated DNA purification (Mijatovic et al, 2005). Paramagnetic-particle handling system usually used to process the sample in large amount and provide consistent yield and purity as there is no detectable cross-contamination between samples. The whole extraction process reported takes about 20 min. There are three steps are needed in extraction using paramagnetic-particles. It start with the addition of liquid samples to reagent cartridge, next, place the reagent cartridges into the machine and lastly to press the start button on the machine. At the last step, the desired DNA is then being eluted into elution buffer (Okamoto et al., 2007). Another example of automated system that is flexible and efficient for extraction of nucleic acids and proteins has been introduced by using various starting materials or sample and those sample can be processed by using this system at once using the same machine and extraction protocol. This system was designed not only for small scale sample and also for medium scale sample. In this system, the purification step using the benefit of the surface functionalized paramagnetic particles to adsorb the isolated nucleic acid (Okamoto et al., 2007). It is reported that the flexibility of this system allows the extraction of nucleic acid up to twelve samples simultaneously and only takes about 20 to 40 min to complete, depending on the application. These kits useful in extracting not only genomic DNA but also cellular RNA, viral and bacterial nucleic acids. As conclusion, automating nucleic acid extraction processes are potentially beneficial for a number of reasons, including reduced working time, decreased labor costs, increasing worker safety and in the midst provides opportunity in increasing reproducibility and quality of results (Boyd, 2002). Besides, it is a key solution to increase the laboratory efficiency. Future directions Now-a-days, the extraction protocol has been standardized and can be easily performed by everyone that gives great benefit and save time. Even though, there were lots of extraction kits are available that does not require tedious lab-based extraction, still there are lots of drawbacks to be improved. Biomolecules extraction for example is the first step that needs to be performed for the downstream analysis or manipulation processes. The liquid handling requirement is the most challenging aspect in extraction process. Therefore, an automatic system must include not only automatic equipment for each extraction step, but also equipment for automating the transfer of liquid between machines. This might reduce the consequent of doing mistakes that will lead to low purity result (Wallace, 1987). Automation is currently highly aided in increasing the throughput and improving the reliability of the process, but these systems are still designed for the use in laboratory environments, which mean in a small scale. Some of the nucleic acid extraction systems that are available in the market, suitable for large scale and to process the higher number of samples, currently require manual pre-processing stages by laboratory staff with technical expertise (Goedecke et al., 2004). Therefore, the urge for robotic workstations in large scale nucleic acid extraction should be fulfill with a fully automated process. A combination of all-in-one biomolecules extraction solution and method with fully automated extraction system can be a prospective invention in the future. There is also need that the purification of DNA, RNA or protein from various organisms can be performed simultaneously using the same type of extraction system with just a single extraction method. It is often inconvenient that targeted biomolecules sample from an animal, plant or even a clinical sample must be sent to the laboratory for it to be extracted and analyzed separately. Current technique requires different protocols that lead time consuming and will eventually destroyed the sample. For some precious sample, such as clinical specimen need to be refrigerated and transfer to the expertise laboratory to be processed, will sometimes reduce the yield of final result. Hence, a portable biomolecules extraction system that could be perform anywhere without the need of expertise really in demand as its brings several advantages such as reduced labor, reduced waste and increased speed of extracting process (Thomsin, 2007). The combination of portable extraction system with DNA, RNA, or protein analyzer can be build up in the future to help researchers in reducing working time and increasing the work efficiency. Thus, continued improvement in miniaturization will be the future trend of robotic automation in the laboratory (Thomsin, 2007). Besides, this automation system can be implemented at relatively low cost, improving the turnaround times and also reduce the labor costs. Introduction to microfluidic Other than that, the manipulation of fluids in channels with dimensions of tens of micrometers, microfluidics has emerged as a distinct new field. Microfluidics has the potential to influence subject areas from chemical synthesis and biological analysis to optics and information technology. Microfluidic is the science and technology of systems that process or manipulate small (10–9 to 10–18 liters) amounts of fluids, using channels with dimensions of tens to hundreds of micrometers. The main applications of microfluidic technologies have been in analysis as they offer a number of useful capabilities such as the ability to use very small quantities of samples and reagents, carry out separations and detections with high resolution and sensitivity, low cost, short times for analysis and small footprints for the analytical devices (Cho et al., 2007). Now-a-days, microfluidics technology not only based on photolithography, but also association of photolithography in silicon microelectronics and in micro electromechanical systems (MEMS) that would be directly applicable to microfluidics. Beginning work in fluidic microsystems uses silicon and glass, but these materials have largely been displaced by plastics. This is due to unnecessary or inappropriate fabricated device in glass and silicon used for analysis of biological samples in water. Silicon, on the other hand, is expensive and opaque to visible and ultraviolet light, thus cannot be used with conventional optical methods of detection. It is easier to fabricate the components required for micro analytical systems, especially pumps and valves in elastomers than in rigid materials. Thus, microfluidic devices in exploratory research have been carried out in a polymer which is called as poly (dimethylsiloxane), or PDMS. The properties of PDMS are entirely distinct from those of silicon (Sia & Kricka, 2008). PDMS is an optically transparent and soft elastomer. Microelectronic technologies however, been indispensable for the development of microfluidics, and as the field has developed, glass, steel and silicon have again emerged as materials which to build specialized systems that require chemical and thermal stability (Daar et al., 2002). Figure 1 shows Scheme describing rapid prototyping of microfluidic systems. A system of channels is designed in a CAD program. A commercial printer uses the CAD file to produce a high-resolution transparency (~5000 dpi). (a) This transparency is used as a photomask in contact photolithography to produce a master. A master consists of a positive relief of photoresist on a silicon wafer and serves as a mold for PDMS. (b) Liquid PDMS pre-polymer is poured over the master and cured for 1 h at 70°C. (c) The PDMS replica is peeled from the master. (d) The replica is sealed to a flat surface to enclose the channels. The overall process takes ~24 h. (McDonald & Whitesides, 2002). Microfluidic for DNA extraction A microfluidic system must have a series of generic components from a method of introducing reagents and samples, methods for moving the fluids around on the chip, combining and mixing the fluids until various other devices such as detectors for most micro analytical work, and components for purification of products for systems used in synthesis. The field has been centered on demonstrating concepts for these components. Two particularly important contributions have been the development of soft lithography in PDMS as a method for fabricating prototype devices, and the development of a simple method of fabricating pneumatically activated valves, mixers and pumps on the basis of soft-lithographic procedures (Gravesen et al., 1993; Dolnik & Jovanovich, 2000). These methods have made it possible to fabricate prototype devices that test new ideas in a time period much shorter than that which could be achieved using silicon technology which is non specializedthat would consume longer period of time. Figure 2 shows an example of the chip that was used for the extraction experiments. The microchannels have been milled directly into a polycarbonate substrate (blank DVD). To illustrate the extraction process DI water dyed with ink has been injected into the chip (outlet port 4a is blocked and therefore filled with air). The different colours denote the different buffer solutions used for the extraction process. Red: lysis and binding buffer including the cell sample and the magnetic beads, blue: washing solution and green: elution buffer. Since in the last century, the miniaturization of electronic devices or microelectronics has been regarded as the most significant enabling technology in human history. With the integrated circuits and progress in information processing, microelectronics has revolutionized the way we work, live and play. Miniaturization concepts have recently been brought into the fluidics since the introduction by Manz et al., at the 5th International conference on Solid-State Sensors and Actuators (Transducers ‘89): microfluidics, which appeared as the name for the new research discipline dealing with transport phenomena and fluid-based devices at microscopic length scales (Manz et al, 1990). One of the most impressive developments of microfluidics in life sciences is ‘Bed-side analyses’ or ‘Point-of-Care testing (POCT)’ such as glucose test meter, pregnancy strip test and others, which is defined as diagnostic testing at or near the site of patient care to make the test convenient and immediate (Sia & Kricka, 2008). Patients can now receive the testing result within minutes. Such devices can be used in hospitals, at a doctor office or simply by patients themselves at home without any professional knowledge or particular skill. Thus, currently, miniaturized devices are likely to impact economy and improve public health significantly (Daar et al, 2002 Other than contributed in biomedicine application, microfluidic also being applied in biotechnology application such as in continuous DNA extraction. The detection of DNA and its variation is critical for many fields, including clinical and veterinary diagnostics, industrial and environmental testing, agricultural researches and forensic science. Disease diagnosis and prognosis are based on effective detection of chronic disease conditions, such as in cancer or contagious disease such as HIV and genetic markers. However, DNA analysis from original specimens is a complex process involving multiple chemical compositions as well as multistep reactions. Conventionally this procedure is time consuming, labor intensive and contamination prone, it is not compatible with high throughput or field testing requirement. To integrate the automatic sample pretreatment functions will reduce reagent consuming, assay time and contamination risk. More importantly, it will not only affect the efficiency and reliability but also determine the feasibility of a final product. In the past decade, about 3,000 papers published about on chip DNA tests. Almost all of them need off-chip sample preparation and reagent handling. A full function system with sample-in–answer-out capability is still rare (Easley et al., 2006). By using microfluidic DNA extraction can be performed anywhere and by anyone without the need of professional assistance. The concentration of DNA analyte in the test samples is usually not yet high enough for direct detection. Therefore, DNA amplification is a required step to raise the concentration of the target sequence. There is many articles and review paper regarding combining the continuous DNA extraction using microfluidic together with DNA amplification. The integration of extraction and polymerase chain reaction (PCR) in the same device becoming the future revolution in POTC (Dineva et al, 2007). A B Figure 3 shows the scheme of mixer construction. Adapted from [219]. (A) Schematic picture showing the PDMS active mixer design and construction. The overall chip size is 3 cm by 1.5 cm. (B) Schematic of integrated device with two liquid samples and electrophoresis gel present. As conclusion, the development of microfluidics still need several steps to go. A number of factors suggest that there are many early-stage applications of microsystems containing fluids, including the exploration of fluidic optics and cells, the development of new types of organic synthesis in small-channel systems, the continuing development of technologies based on large arrays of detectors and on high-throughput screening, the fabrication of microrobotic systems using hydraulic systems based on microfluidics, other fluidic versions of MEMS, and work on biomimetic systems with microfluidic components. Different types of micro/nano-fluidic technologies have facilitated DNA purification, amplification and detection to be integrated into one chip which combine the advantages of small sizes, much shorter reaction times, less manual operation and reduced cost. Successful DNA amplification and detection on chip depends on the optimization of several parameters, which is a cumbersome task due to many variables (conditions and components) typically involved and requires comprehensive knowledge of multi-subject intersecting molecular biology, chemistry, physics, mechanics and micro/nano-fabrication technologies. Conclusion DNA plays many roles now-a-days, not only for the interest of the researcher but it is also important for the phylogenic tree to trace the species of new things. For forensic applications, to trace criminals and also for medical purposes with hereditary and unknown diseases. Thus, DNA extraction process has attract the biggest interest as re-cap back when the first DNA isolation was successfully done by Friedrich Miescher in 1869 and the initial DNA extraction developed from density gradient centrifugation strategies by Meselson and Stahl in 1958, many techniques for biomolecules purification has been developed afterwards. From the phenol-chloroform extraction to the column-technology has widely used in nucleic acids extraction. Biomolecules extraction has helped researchers and scientists in manipulating subsequent molecular biology analysis in order to have a better understanding about the biological materials. The automated nucleic acid extraction system has been developed due to the influence of rapid growth of automation technology. Automating nucleic acid extraction process is potentially beneficial for a number of reasons including to reduce working time, decrease labor costs, increase worker safety and at the same time provides opportunity in increasing reproducibility and quality of results. However, improvement with the currently available instruments needs to be conducted all the time. In the meantime, an all-in-one biomolecules extraction system, or the invention of a miniature and portable extraction system can become a prospective development in the future. References Arnold, T. 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