The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher.[1]DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR,[2]sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.
This process can be done in several ways, depending on the type of the sample and the downstream application,[3] the most common methods are: mechanical, chemical and enzymatic lysis, precipitation, purification, and concentration. The specific method used to extract the DNA, such as phenol-chloroform extraction, alcohol precipitation, or silica-based purification.[4]
For the chemical method, many different kits are used for extraction, and selecting the correct one will save time on kit optimization and extraction procedures. PCR sensitivity detection is considered to show the variation between the commercial kits.[5]
There are many different methods for extracting DNA, but some common steps include:
Lysis: This step involves breaking open the cells to release the DNA. For example, in the case of bacterial cells, a solution of detergent and salt (such as SDS) can be used to disrupt the cell membrane and release the DNA. For plant and animal cells, mechanical or enzymatic methods are often used.
Precipitation: Once the DNA is released, proteins and other contaminants must be removed. This is typically done by adding a precipitating agent, such as alcohol (such as ethanol or isopropanol), or a salt (such as ammonium acetate). The DNA will form a pellet at the bottom of the solution, while the contaminants will remain in the liquid.
Purification: After the DNA is precipitated, it is usually further purified by using column-based methods. For example, silica-based spin columns can be used to bind the DNA, while contaminants are washed away. Alternatively, a centrifugation step can be used to purify the DNA by spinning it down to the bottom of a tube.
Concentration: Finally, the amount of DNA present is usually increased by removing any remaining liquid. This is typically done by using a vacuum centrifugation or a lyophilization (freeze-drying) step.
Some variations on these steps may be used depending on the specific DNA extraction protocol. Additionally, some kits are commercially available that include reagents and protocols specifically tailored to a specific type of sample.[6]
What does it deliver?
DNA extraction is frequently a preliminary step in many diagnostic procedures used to identify environmental viruses and bacteria and diagnose illnesses and hereditary diseases. These methods consist of, but are not limited to:
Fluorescence In Situ Hybridization (FISH) technique was developed in the 1980s. The basic idea is to use a nucleic acid probe to hybridize nuclear DNA from either interphase cells or metaphase chromosomes attached to a microscopic slide. It is a molecular method used, among other things, to recognize and count particular bacterial groupings.[1]
To recognize, define, and quantify the geographical and temporal patterns in marine bacterioplankton communities, researchers employ a technique called terminal restriction fragment length polymorphism (T-RFLP).
Sequencing: Whole or partial genomes and other chromosomal components, ended for comparison with previously published sequences.
The solution is treated with a concentrated salt solution (saline) to make debris such as broken proteins, lipids, and RNA clump together.
Centrifugation of the solution, which separates the clumped cellular debris from the DNA.
DNA purification from detergents, proteins, salts, and reagents is used during the cell lysis step. The most commonly used procedures are:
Ethanol precipitation usually by ice-cold ethanol or isopropanol. Since DNA is insoluble in these alcohols, it will aggregate together, giving a pellet upon centrifugation. Precipitation of DNA is improved by increasing ionic strength, usually by adding sodium acetate.
Phenol–chloroform extraction in which phenoldenatures proteins in the sample. After centrifugation of the sample, denatured proteins stay in the organic phase while the aqueous phase containing nucleic acid is mixed with chloroform to remove phenol residues from the solution.
After isolation, the DNA is dissolved in a slightly alkaline buffer, usually in a TE buffer, or in ultra-pure water.
Common chemicals
The most common chemicals used for DNA extraction include:
Detergents, such as SDS or Tween-20, which are used to break open cells and release the DNA.
Protease enzymes, such as Proteinase K, which are used to digest proteins that may be binding to the DNA.
Phenol and chloroform, which are used to separate the DNA from other cellular components.
Ethanol or isopropanol, which are used to precipitate the DNA.
Salt, such as NaCl, which is often used to help dissolve the DNA and maintain its stability.
EDTA, which is used to chelate the metals ions that can damage the DNA.
Tris-HCL, which is used to maintain the pH at the optimal condition for DNA extraction.
Method selection
Some of the most common DNA extraction methods include organic extraction, Chelex extraction, and solid phase extraction.[8] These methods consistently yield isolated DNA, but they differ in both the quality and the quantity of DNA yielded. When selecting a DNA extraction method, there are multiple factors to consider, including cost, time, safety, and risk of contamination.
Organic extraction involves the addition of incubation in multiple different chemical solutions;[8] including a lysis step, a phenol-chloroform extraction, an ethanol precipitation, and washing steps. Organic extraction is often used in laboratories because it is cheap, and it yields large quantities of pure DNA. Though it is easy, there are many steps involved, and it takes longer than other methods. It also involves the unfavorable use of the toxic chemicals phenol and chloroform, and there is an increased risk of contamination due to transferring the DNA between multiple tubes.[9] Several protocols based on organic extraction of DNA were effectively developed decades ago,[10] though improved and more practical versions of these protocols have also been developed and published in the last years.[11]
The chelex extraction method involves adding the Chelex resin to the sample, boiling the solution, then vortexing and centrifuging it. The cellular materials bind to the Chelex beads, while the DNA is available in the supernatant.[9] The Chelex method is much faster and simpler than organic extraction, and it only requires one tube, which decreases the risk of DNA contamination. Unfortunately, Chelex extraction does not yield as much quantity and the DNA yielded is single-stranded, which means it can only be used for PCR-based analyses and not for RFLP.[9]
Solid phase extraction such as using a spin-column-based extraction method takes advantage of the fact that DNA binds to silica. The sample containing DNA is added to a column containing a silica gel or silica beads and chaotropic salts. The chaotropic salts disrupt the hydrogen bonding between strands and facilitate the binding of the DNA to silica by causing the nucleic acids to become hydrophobic. This exposes the phosphate residues so they are available for adsorption.[12] The DNA binds to the silica, while the rest of the solution is washed out using ethanol to remove chaotropic salts and other unnecessary constituents.[8] The DNA can then be rehydrated with aqueous low-salt solutions allowing for elution of the DNA from the beads.
This method yields high-quality, largely double-stranded DNA which can be used for both PCR and RFLP analysis. This procedure can be automated[9] and has a high throughput, although lower than the phenol-chloroform method. This is a one-step method i.e. the entire procedure is completed in one tube. This lowers the risk of contamination making it very useful for the forensic extraction of DNA. Multiple solid-phase extraction commercial kits are manufactured and marketed by different companies; the only problem is that they are more expensive than organic extraction or Chelex extraction.
Special types
Specific techniques must be chosen for the isolation of DNA from some samples. Typical samples with complicated DNA isolation are:
archaeological samples containing partially degraded DNA, see ancient DNA[13]
samples containing inhibitors of subsequent analysis procedures, most notably inhibitors of PCR, such as humic acid from the soil, indigo and other fabric dyes or haemoglobin in blood
samples from microorganisms with thick cellular walls, for example, yeast
samples containing mixed DNA from multiple sources
Extrachromosomal DNA is generally easy to isolate, especially plasmids may be easily isolated by cell lysis followed by precipitation of proteins, which traps chromosomal DNA in insoluble fraction and after centrifugation, plasmid DNA can be purified from soluble fraction.
A Hirt DNA Extraction is an isolation of all extrachromosomal DNA in a mammalian cell. The Hirt extraction process gets rid of the high molecular weight nuclear DNA, leaving only low molecular weight mitochondrial DNA and any viral episomes present in the cell.
A diphenylamine (DPA) indicator will confirm the presence of DNA. This procedure involves chemical hydrolysis of DNA: when heated (e.g. ≥95°C) in acid, the reaction requires a deoxyribose sugar and therefore is specific for DNA. Under these conditions, the 2-deoxyribose is converted to w-hydroxylevulinyl aldehyde, which reacts with the compound, diphenylamine, to produce a blue-colored compound. DNA concentration can be determined by measuring the intensity of absorbance of the solution at the 600nm with a spectrophotometer and comparing to a standard curve of known DNA concentrations.
Measuring the intensity of absorbance of the DNA solution at wavelengths 260 nm and 280 nm is used as a measure of DNA purity. DNA can be quantified by cutting the DNA with a restriction enzyme, running it on an agarose gel, staining with ethidium bromide (EtBr) or a different stain and comparing the intensity of the DNA with a DNA marker of known concentration.
Using the Southern blot technique, this quantified DNA can be isolated and examined further using PCR and RFLP analysis. These procedures allow differentiation of the repeated sequences within the genome. It is these techniques which forensic scientists use for comparison, identification, and analysis.
High-molecular-weight DNA extraction method
In this method, plant nuclei are isolated by physically grinding tissues and reconstituting the intact nuclei in a unique Nuclear Isolation Buffer (NIB). The plastid DNAs are released from organelles and eliminated with an osmotic buffer by washing and centrifugation. The purified nuclei are then lysed and further cleaned by organic extraction, and the genomic DNA is precipitated with a high concentration of CTAB. The highly pure, high molecular weight gDNA is extracted from the nuclei, dissolved in a high pH buffer, allowing for stable long-term storage.[14]
DNA storage
DNA storage is an important aspect of DNA extraction projects as it ensures the integrity and stability of the extracted DNA for downstream applications.[15]
One common method of DNA storage is ethanol precipitation, which involves adding ethanol and a salt, such as sodium chloride or potassium acetate, to the extracted DNA to precipitate it out of solution. The DNA is then pelleted by centrifugation and washed with 70% ethanol to remove any remaining contaminants. The DNA pellet is then air-dried and resuspended in a buffer, such as Tris-EDTA (TE) buffer, for storage.
Another method is freezing the DNA in a buffer such as TE buffer, or in a cryoprotectant such as glycerol or DMSO, at -20 or -80 degrees Celsius. This method preserves the integrity of the DNA and slows down the activity of any enzymes that may degrade it.
It's important to note that the choice of storage buffer and conditions will depend on the downstream application for which the DNA is intended. For example, if the DNA is to be used for PCR, it may be stored in TE buffer at 4 degrees Celsius, while if it is to be used for long-term storage or shipping, it may be stored in ethanol at -20 degrees Celsius. The extracted DNA should be regularly checked for its quality and integrity, such as by running a gel electrophoresis or spectrophotometry. The storage conditions should be also noted and controlled, such as the temperature and humidity.
It's also important to consider the long-term stability of the DNA and the potential for degradation over time. The extracted DNA should be stored for as short a time as possible, and the conditions for storage should be chosen to minimize the risk of degradation.
In general, the extracted DNA should be stored under the best possible conditions to ensure its stability and integrity for downstream applications.
Quality control
There are several quality control techniques used to ensure the quality of extracted DNA, including:[16]
Spectrophotometry: This is a widely used method for measuring the concentration and purity of a DNA sample. Spectrophotometry measures the absorbance of a sample at different wavelengths, typically at 260nm and 280nm. The ratio of absorbance at 260nm and 280nm is used to determine the purity of the DNA sample.[17]
Gel electrophoresis: This technique is used to visualize and compare the size and integrity of DNA samples. The DNA is loaded onto an agarose gel and then subjected to an electric field, which causes the DNA to migrate through the gel. The migration of the DNA can be visualized using ethidium bromide, which intercalates into the DNA and fluoresces under UV light.[18]
Fluorometry: Fluorometry is a method to determine the concentration of nucleic acids by measuring the fluorescence of the sample when excited by a specific wavelength of light. Fluorometry uses dyes that specifically bind to nucleic acids and have a high fluorescence intensity.
PCR: Polymerase Chain Reaction (PCR) is a technique that amplifies a specific region of DNA, it is also used as a QC method by amplifying a small fragment of the DNA, if the amplification is successful, it means the extracted DNA is of good quality and it's not degraded.
Qubit Fluorometer: The Qubit Fluorometer is an instrument that uses fluorescent dyes to measure the concentration of DNA and RNA in a sample. It is a quick and sensitive method that can be used to determine the concentration of DNA samples.[16]
Bioanalyzer: The bioanalyzer is an instrument that uses electrophoresis to separate and analyze DNA, RNA, and protein samples. It can provide detailed information about the size, integrity, and purity of a DNA sample.
Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.
A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells. Most lysis buffers contain buffering salts and ionic salts to regulate the pH and osmolarity of the lysate. Sometimes detergents are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells.
Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.
Salting out is a purification technique that utilizes the reduced solubility of certain molecules in a solution of very high ionic strength. Salting out is typically used to precipitate large biomolecules, such as proteins or DNA. Because the salt concentration needed for a given protein to precipitate out of the solution differs from protein to protein, a specific salt concentration can be used to precipitate a target protein. This process is also used to concentrate dilute solutions of proteins. Dialysis can be used to remove the salt if needed.
Ammonium sulfate precipitation is one of the most commonly used methods for large and laboratory scale protein purification and fractionation that can be used to separate proteins by altering their solubility in the presence of a high salt concentration.
Phenol extraction is a laboratory technique that purifies nucleic acid samples using a phenol solution. Phenol is common reagent in extraction because its properties allow for effective nucleic acid extraction, particularly as it strongly denatures proteins, it is a nucleic acid preservative, and it is immiscible in water.
A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. It is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.
Ethanol precipitation is a method used to purify and/or concentrate RNA, DNA, and polysaccharides such as pectin and xyloglucan from aqueous solutions by adding salt and ethanol as an antisolvent.
Differential extraction refers to the process by which the DNA from two different types of cells can be extracted without mixing their contents. The most common application of this method is the extraction of DNA from vaginal epithelial cells and sperm cells from sexual assault cases in order to determine the DNA profiles of the victim and the perpetrator. Its success is based on the fact that sperm cells pack their DNA using protamines which are held together by disulfide bonds. The protamines sequester DNA from spermatozoa, making it more resilient to DNA extraction than DNA from epithelial cells.
Electroelution is a method used to extract a nucleic acid or a protein sample from an electrophoresis gel by applying a negative current in the plane of the smallest dimension of the gel, drawing the macromolecule to the surface for extraction and subsequent analysis. For example, electroblotting and preparative native PAGE are based upon the same principle.
Alkaline lysis or alkaline extraction is a method used in molecular biology to isolate plasmid DNA from bacteria.
Acid guanidinium thiocyanate-phenol-chloroform extraction is a liquid–liquid extraction technique in biochemistry and molecular biology. It is widely used for isolating RNA. This method may take longer than a column-based system such as the silica-based purification, but has higher purity and the advantage of high recovery of RNA. Furthermore, an RNA column is typically unsuitable for purification of short RNA species, such as siRNA, miRNA and tRNA.
DNA separation by silica adsorption is a method of DNA separation that is based on DNA molecules binding to silica surfaces in the presence of certain salts and under certain pH conditions.
TRIzol is a widely used chemical solution used in the extraction of DNA, RNA, and proteins from cells. The solution was initially used and published by Piotr Chomczyński and Nicoletta Sacchi in 1987.
RNA extraction is the purification of RNA from biological samples. This procedure is complicated by the ubiquitous presence of ribonuclease enzymes in cells and tissues, which can rapidly degrade RNA. Several methods are used in molecular biology to isolate RNA from samples, the most common of these is guanidinium thiocyanate-phenol-chloroform extraction. The filter paper based lysis and elution method features high throughput capacity.
Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.
Boom method is a solid phase extraction method for isolating nucleic acid from a biological sample. This method is characterized by "absorbing the nucleic acids (NA) to the silica beads".
Phenol–chloroform extraction is a liquid-liquid extraction technique in molecular biology used to separate nucleic acids from proteins and lipids.
Small RNA sequencing is a type of RNA sequencing based on the use of NGS technologies that allows to isolate and get information about noncoding RNA molecules in order to evaluate and discover new forms of small RNA and to predict their possible functions. By using this technique, it is possible to discriminate small RNAs from the larger RNA family to better understand their functions in the cell and in gene expression. Small RNA-Seq can analyze thousands of small RNA molecules with a high throughput and specificity. The greatest advantage of using RNA-seq is represented by the possibility of generating libraries of RNA fragments starting from the whole RNA content of a cell.
Solid-phase reversible immobilization, or SPRI, is a method of purifying nucleic acids from solution. It uses silica- or carboxyl-coated paramagnetic beads, which reversibly bind to nucleic acids in the presence of polyethylene glycol and a salt. A common application of SPRI technology is purifying samples of DNA amplified by PCR for sequencing reactions:.
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