They also provided only small quantities of DNA. Today there are many specialised extraction methods. These are generally either solution-based or column-based. The extraction of DNA has become much easier with the emergence of commercial kits and the automation of the process. Such changes have both sped up production and increased the yield of DNA.
The ability to extract DNA is of primary importance to studying the genetic causes of disease and for the development of diagnostics and drugs. It is also essential for carrying out forensic science, sequencing genomes, detecting bacteria and viruses in the environment and for determining paternity. Respond to or comment on this page on our feeds on Facebook , Instagram or Twitter. However, he lacked the skills to communicate and promote what he had found to the wider scientific community.
Ever the perfectionist, he hesitated for long periods of time between experiments before he published his results in Before then he primarily discussed his findings in private letters to his friends. For many years, scientists continued to believe that proteins were the molecules that held all of our genetic material. Surely, one type of molecule could not account for all the variation seen within species?
Albrecht Kossel was a German biochemist who made great progress in understanding the basic building blocks of nuclein. Albrecht Kossel isolated the five nucleotide bases that are the building blocks of DNA and RNA: adenine, cytosine, guanine, thymine and uracil.
In Albrecht identified nuclein as a nucleic acid and provided its present chemical name, deoxyribonucleic acid DNA. In the early s, the work of Gregor Mendel was rediscovered and his ideas about inheritance began to be properly appreciated.
As a result, a flood of research began to try and prove or disprove his theories of how physical characteristics are inherited from one generation to the next.
In the middle of the nineteenth century, Walther Flemming, an anatomist from Germany, discovered a fibrous structure within the nucleus of cells. By observing this chromatin, Walther correctly worked out how chromosomes separate during cell division, also known as mitosis.
Walter Sutton and Theodor Boveri first presented the idea that the genetic material passed down from parent to child is within the chromosomes. The chromosome theory of inheritance was developed primarily by Walter Sutton and Theodor Boveri. They first presented the idea that the genetic material passed down from parent to child is within the chromosomes. Their work helped explain the inheritance patterns that Gregor Mendel had observed over a century before. Interestingly, Walter Sutton and Theodor Boveri were actually working independently during the early s.
Walter studied grasshopper chromosomes, while Theodor studied roundworm embryos. However, their work came together in a perfect union, along with the findings of a few other scientists, to form the chromosome theory of inheritance.
Walter Sutton left and Theodor Boveri right worked independently to come up with the chromosome theory of inheritance. Image credit: Wikimedia Commons. DNA, RNA, and protein can be isolated from any biological material such as living or conserved tissues, cells, virus particles, or other samples for analytical or preparative purposes [ 1 ].
Two categories that involved in purifying DNA include the isolation of recombinant DNA constructs such as plasmids or bacteriophage and the isolation of chromosomal or genomic DNA from prokaryotic or eukaryotic organisms [ 2 ].
Generally, successful nucleic acid purification required four important steps: effective disruption of cells or tissue; denaturation of nucleoprotein complexes; inactivation of nucleases, for example, RNase for RNA extraction and DNase for DNA extraction; away from contamination [ 2 ].
Quality and also integrity of the isolated nucleic acid will directly affect the results of all succeeding scientific research [ 4 ]. On the other hand, RNA is an unstable molecule and has a very short half-life once extracted from the cell or tissues [ 5 ].
Special care and precautions are required for RNA isolation as it is susceptible to degradation [ 3 , 6 ]. RNA is especially unstable due to the ubiquitous presence of RNases which are enzymes present in blood, all tissues, as well as most bacteria and fungi in the environment [ 3 , 5 ].
RNA extraction relies on good laboratory technique and RNase-free technique. RNAse is heat-stable and refolds following heat denaturation. They are difficult to inactivate as they do not require cofactors [ 2 ]. Purification of protein is one of the most important parts in protein research to understand their function, as they may partly or completely be involved in any DNA synthesis activity.
Protein purification is required to determine its unique characteristics, including size, charge, shape, and function [ 7 ].
Cell-based extraction is the starting step for almost all protein purification. Protein can be extracted by a few methods such as detergent lysis, shearing force, treatment with low ionic salt salting out , and rapid changes in pressure, which aimed to weaken and break the membranes surrounding the cell to allow proteins to escape [ 7 ]. Some factors should be considered when handling proteins. Buffer condition is one of the major factors that need to be considered.
Specific buffer conditions are recommended to be maintained because of the sensitivity of proteins toward environmental pH changes [ 4 ]. The purity of water will affect the yield of end products as unpurified water contains a lot of microorganisms or proteases that will result in protein degradation [ 4 ]. Protein inhibitor, which may exist in solution or buffers, causes the hydrolyzation of proteins.
They solubilize the membrane protein and are amphiphatic molecules which form micelles with the hydrophilic head of proteins [ 4 ]. Reducing agents will be added into solution or buffer for protein extraction and purification to avoid the lost of activity of proteins or enzymes which is caused by oxidization.
Storage of proteins is important as the half-life of protein is commonly dependent on the storage temperature [ 4 ]. The purification of protein requires specific assay.
A quick and easy assay method must be known for protein purification so that a known molecular weight, specific affinity, or immunoaffinity of nonenzymatic protein of interest can be detected using appropriate method [ 7 ]. There are several methods commonly used in protein purification. They are ion exchange chromatography, gel filtration, affinity chromatography and gel electrophoresis [ 4 ]. He hoped to solve the fundamental principles of life, to determine the chemical composition of cells.
He tried to isolate cells from lymph nodes for his experiment but the purity of lymphocytes was hard and impossible to be obtained in sufficient quantities.
Therefore, he switched to leucocytes, where he obtained them from the pus on collected surgical bandages. Initially, Miescher focused on the various type of protein that make up the leukocytes and showed that proteins were the main components of the cell's cytoplasm.
During his tests, he noticed that a substance precipitated from the solution when acid was added and dissolved again when alkali was added. This was, for the first time he had obtained a crude precipitate of DNA. To separate DNA from the proteins in his cell extracts, Miescher developed new protocol to separate the cells' nuclei from cytoplasm and then isolated DNA.
However, his first protocol failed to yield enough material to continue with further analysis. In the eighteenth century, proteins were known as a distinct class of biological molecules by Antoine Fourcroy and others. They distinguished this molecule by its ability to coagulate under treatment with heat or acid. However, the first description of protein was carried out by Gerhardus Johannes Mulder, a Dutch chemist, in [ 9 ].
His studies on the composition of animal substances, mainly fibrin, albumin, and gelatin, showed the presence of carbon, hydrogen, oxygen, and nitrogen [ 9 ]. Most of the early studies focused on proteins that could be purified in large quantities. For example, blood, egg white and various toxins.
Most of the proteins are hard to purify in more than milligram quantities even with today's highly advanced methods. A majority of techniques for protein purification were developed in a project led by Edwin Joseph Cohn, a protein scientist, during World War II.
He was responsible for purifying blood and worked out the techniques for isolating the serum albumin fraction of blood plasma, which is important in maintaining the osmotic pressure in the blood vessels, which help keep soldier alive [ 10 ].
After the fated event where Miescher managed to obtain DNA from cell, many others have followed suit which lead to further advancement in the DNA isolation and purification protocol. The initial routine laboratory procedures for DNA extraction were developed from density gradient centrifugation strategies. Meselson and Stahl used this method in to demonstrate semiconservative replication of DNA [ 3 ].
Later procedures made use of the differences in solubility of large chromosomal DNA, plasmids, and proteins in alkaline buffer [ 3 ]. Generally, they are divided into solution-based or column-based protocols. Most of these protocols have been developed into commercial kits that ease the biomolecules extraction processes. Salt is the common impurity in nucleic acid samples.
It has always been required to be removed from nucleic acid samples before any downstream processes and analysis can be done. The general steps of nucleic acid purification include cell lysis, which disrupts the cellular structure to create a lysate, inactivation of cellular nucleases such as DNase and RNase, and separation of desired nucleic acid from cell debris [ 2 ]. Organic solvent—phenol-chloroform extraction is one of the examples, which is widely used in isolating nucleic acid.
Although phenol, a flammable, corrosive, and toxic carbolic acid can denature proteins rapidly, it does not completely inhibit RNAse activity [ 12 ]. This problem can be solved by using a mixture of phenol: chloroform: isoamyl alcohol Proteins, lipids, carbohydrates, and cell debris are removed through extraction of the aqueous phase with the organic mixture of phenol and chloroform [ 12 , 13 ]. A biphasic emulsion forms when phenol and chloroform are added.
The hydrophobic layer of the emulsion will then be settled on the bottom and the hydrophilic layer on top by centrifugation [ 3 ]. The upper phase which contained DNA is collected and DNA can be precipitated from the supernatant by adding ethanol or isopropanol in 2 : 1 or 1 : 1 ratios and high concentration of salt [ 3 ].
The use of guanidinium isothiocyanate in RNA extraction was first mentioned by Ulrich et al. The method was laborious. Guanidinium thiocyanate is a chaotropic agent used in protein degradation. The principle of this single-step technique is that RNA is separated from DNA after extraction with acidic solution consisting guanidinium thiocyanate, sodium acetate, phenol, and chloroform [ 13 ]. In the acidic conditions, total RNA will remain in the upper aqueous phase of the whole mixture, while DNA and proteins remain in the interphase or lower organic phase.
Recovery of total RNA is then done by precipitation with isopropanol [ 12 ]. Alkaline lysis has been used to isolate plasmid DNA and E. It works well with all strains of E. The principle of the method is based on selective alkaline denaturation of high molecular weight chromosomal DNA while covalently closed circular DNA remains double stranded [ 14 ]. Bacterial proteins, broken cell walls, and denatured chromosomal DNA enmeshed into large complexes that are coated with dodecyl sulfate.
Plasmid DNA can be recovered from the supernatant after the denatured material has been removed by centrifugation. For plant extraction, the initial step that needs to be done is to grind the sample after freezing it with liquid nitrogen. The purpose of doing this step is to break down cell wall material of sample and allow access to nucleic acid while harmful cellular enzymes and chemicals remain inactivated.
After grinding the sample, it can be resuspended in a suitable buffer such as CTAB. Cetyltrimethylammonium bromide CTAB is a nonionic detergent that can precipitate nucleic acids and acidic polysaccharides from low ionic strength solutions [ 15 ]. Meanwhile, proteins and neutral polysaccharides remain in solution under these conditions. In solutions of high ionic strength, CTAB will not precipitate nucleic acids and forms complexes with proteins. CTAB is therefore useful for purification of nucleic acid from organisms which produce large quantities of polysaccharides such as plants and certain Gram-negative bacteria [ 15 ].
This method also uses organic solvents and alcohol precipitation in later steps [ 12 ]. Insoluble particles are removed through centrifugation to purify nucleic acid. Soluble proteins and other material are separated through mixing with chloroform and centrifugation. Nucleic acid must be precipitated after this from the supernatant and washed thoroughly to remove contaminating salts.
The purified nucleic acid is then resuspended and stored in TE buffer or sterile distilled water. CsCl gradient centrifugation is a complicated, expensive, and time-consuming method compared to other purification protocols.
It requires large scale bacterial culture. Therefore, it is not suitable for the minipreparation of plasmid DNA [ 4 ]. Nucleic acids can be concentrated by centrifugation in an EtBr-CsCl gradient after alcohol precipitation and resuspension. Intercalation of EtBr alters the swimming density of the molecule in high molar CsCl. Covalently 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 will be reprecipitated with alcohol [ 1 ]. High salt must be added to the chromatography buffer to stabilize the nucleic acid duplexes as only a few dT-A base pairs are formed.
A low-salt buffer is used after nonpolyadenylated RNAs have been washed from the matrix. It can be used when many RNA samples are to be processed, whether radioactive or not. Batch chromatography is carried out with a fine grade of oligo dT cellulose at optimal temperatures for binding and elution [ 15 ].
Solid-phase nucleic acid purification can be found in most of the commercial extraction kits available in market. It allows quick and efficient purification compared to conventional methods [ 16 ]. Many of the problems that are associated with liquid-liquid extraction such as incomplete phase separation can be prevented.
Solid phase system will absorb nucleic acid in the extraction process depending on the pH and salt content of the buffer. The absorption process is based on the following principles: hydrogen-binding interaction with a hydrophilic matrix under chaotropic conditions, ionic exchange under aqueous conditions by means of an anion exchanger, and affinity and size exclusion mechanisms.
Solid-phase purification is normally performed by using a spin column, operated under centrifugal force [ 17 ]. This method can purify nucleic acid rapidly compared to conventional methods. Silica matrices, glass particles, diatomaceous earth, and anion-exchange carriers are examples that have been utilized in solid-phase extraction method as solid support.
Four key steps involved in solid-phase extraction are cell lysis, nucleic acids adsorption, washing, and elution [ 6 ]. The initial step in a solid phase extraction process is to condition the column for sample adsorption. Column conditioning can be done by using a buffer at a particular pH to convert the surface or functional groups on the solid into a particular chemical form.
Next, the sample which has been degraded by using lysis buffer is applied to the column. The desired nucleic acid will absorb to the column with the aid of high pH and salt concentration of the binding solution [ 17 ]. Other compounds, such as protein may have strong specific bond with the column surface as well. These contaminants can be removed in the washing step by using washing buffer containing a competitive agent [ 17 ]. For the elution step, TE buffer or water is introduced to release the desired nucleic acid from the column, so that it can be collected in a purified state [ 17 ].
Normally, rapid centrifugation, vacuum filtration, or column separation is required during the washing and elution steps of purification process. A mixed-bed solid phase nucleic acid extraction and its use in the isolation of nucleic acid have been disclosed [ 18 ]. The mixed-bed solid phases of this invention are the mixtures of at least two different solid phases, can be solid or semisolid, porous or non-porous.
Each solid phase can bind to the target nucleic acid under different solution conditions and release the nucleic acid under similar elution conditions [ 18 ]. The basis for most of the products related to nucleic acid purification is the unique properties of silica matrices for selective DNA binding. Types of silica materials including glass particles, such as glass powder, silica particles, and glass microfibers prepared by grinding glass fiber filter papers, and including diatomaceous earth [ 19 ].
Hydrated silica matrix, which was prepared by refluxing silicon dioxide in sodium hydroxide or potassium hydroxide at a molar ratio of about to for at least about 48 hours, had been introduced in DNA purification. DNA binds to the inorganic matrix and is released in heated water [ 20 ].
The principle of silica matrices purification is based on the high affinity of the negatively charged DNA backbone towards the positively charged silica particles [ 21 ]. Sodium plays a role as a cation bridge that attracts the negatively charged oxygen in the phosphate backbone of nucleic acid [ 22 ]. The DNA is tightly bound, and extensive washing removes all contaminations.
Besides silica matrices, nitrocellulose and polyamide membranes such as nylon matrices are also known to bind with nucleic acids, but with less specificity. These materials are often used as solid-phase nucleic acid transfer and hybridization matrices [ 23 ]. Polyamide matrices are more durable than nitrocellulose and are known to bind nucleic acids irreversibly. Nucleic acids can be immobilized on polyamide matrices in low ionic strength buffer [ 23 ].
Glass particles, powder and beads are useful for nucleic acid purification. For example, DNA isolation from agarose gels involved the use of chaotropic salts to facilitate binding of DNA to common silicate glass, flint glass, and borosilicate glass glass fiber filter. The adsorption of nucleic acid onto the glass substrate occurs most likely based on the mechanism and principle that similar to adsorption chromatography [ 24 ]. Nucleic acid purification can also be done on silica gel and glass mixture [ 19 ].
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. It has been used for filtration and in chromatography and it is useful for the purification of plasmid and other DNA by immobilizing DNA onto its particles in the presence of a chaotropic agent.
The resulting diatomaceous earth-bound DNA is then washed with an alcohol-containing buffer. The alcohol—containing buffer is then discarded and DNA is eluted out in a low salt buffer or in distilled water [ 25 ]. Magnetic separation is a simple and efficient way which is used in purification of nucleic acid nowadays.
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. Often, magnetic carriers with immobilized affinity ligands or prepared from biopolymer showing affinity to the target nucleic acid are used for the isolation process.
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. Materials with a large surface area are preferred to be used in the binding of nucleic acids. Magnetic particulate materials such as beads are more preferable to be a support in isolation process because of their larger binding capacity.
A magnet can be applied to the side of the vessel, which contains the sample mixture for aggregating the particles near the wall of the vessel and pouring away the remainder of the sample [ 26 ]. Particles having magnetic or paramagnetic properties are employed in an invention where they are encapsulated in a polymer such as magnetizable cellulose [ 27 ]. In the presence of certainconcentrations 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.
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