DNA Isolation Methods
DNA isolation methods (Forensic Science)
The isolation of DNA (deoxyribonucleic acid) from biological material can be relatively straightforward, but forensic scientists must consider several factors before commencing. The first of these is the planned subsequent DNA analysis, including whether the DNA can be single-stranded, as for polymerase chain reaction (PCR) analyses, or must be double-stranded, as for restriction fragment length polymorphism (RFLP) testing. The source of the sample (whether blood, semen, hair, or bone) will influence processing choices and may mean that a tissue must be processed in advance (for example, skeletal material may need to be ground). Other considerations include the desired level of DNA cleanliness, the maximization of yield, the minimization of processing steps, the number of samples, the presence of mixtures, and the presence of potential PCR inhibitors (such as iron in blood or humic acid in soil).
Commonly used methods of DNA isolation in forensic laboratories include organic extraction, differential extraction, Chelex, the use of preservative papers, the use of isolation kits, and the use of robotics. All of these involve breaking open cells (lysis) to release DNA, purification to remove unwanted material, and harvesting the DNA for analysis.
Once an extraction method is chosen, the analyst must take steps to prevent contamination. Proper training of laboratory personnel is imperative; disposable gloves and protective clothing should...
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Organic Extraction (Forensic Science)
Organic extractions are widely used in forensic laboratories owing to their general applicability and the purity of the resultant DNA. Following cell lysis (with a proteinase and detergent), undesired materials (such as fats and proteins) are solubilized into an organic solvent such as phenol or chloroform.
Organic extractions can be time-consuming, but the DNA collected is relatively clean, can be used for any type of subsequent analysis, and is amenable to any tissue type. Disadvantages of these methods include lengthy time expenditure and exposure to hazardous chemicals.
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Differential Extraction (Forensic Science)
An expanded organic method is the differential extraction, which is used on samples from sexual assaults, particularly vaginal swabs, which can contain epithelial cells from the victim and sperm from the perpetrator. Differential extractions take advantage of the dissimilar nature of sperm and epithelial cell walls. The sample is first placed in a mild lysis buffer that releases epithelial cell DNA while sperm remain intact. The sperm are pelleted by centrifugation, and the liquid containing epithelial DNA (the nonsperm fraction) is removed and purified organically. The sperm are then lysed under stronger conditions, and this male/sperm fraction is purified. Differential extraction allows enrichment of each fraction by upward of 90 percent, helping to clarify mixture results.
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Chelex Extraction (Forensic Science)
DNA preparation using Chelex (iminodiacetic acid bound to polystyrene beads) has two positive attributes: It is fast and it is easy. The entire procedure is carried out in a single tube and is generally performed on blood and saliva, although other tissues may be considered. The major objective of a Chelex extraction is to bind (chelate) unwanted metals that can inhibit PCR, notably polyvalent cations (such as iron and calcium). The sample is boiled, and upon centrifugation the beads are forced to the bottom of the tube, leaving the DNA in solution ready for quantification and amplification. Because minimal purification steps are involved in this method, the DNA is not pristine (hence it may not amplify or store well). Also, owing to the boiling step, the DNA is single-stranded.
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Preservative Papers (Forensic Science)
One of the simplest methods for extracting DNA is through the use of special papers chemically treated to lyse cells and denature proteins on contact. A liquid sample (blood, saliva) is applied to the paper and dried, then stored at room temperature or used immediately. A small punch of the stained paper is collected, washed, and subjected directly to PCR. Extended sample stability is the major advantage of the use of preservative papers; disadvantages include possible difficulty in manipulating the papers and the fact that this method produces no DNA quantification.
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Commercial DNA Isolation Kits (Forensic Science)
Several companies have developed commercial kits for DNA purification. These tend to be quick (as little as thirty minutes) and easy to use, but they are often expensive. Kits can allow for a large number of samples to be processed simultaneously, and manufacturers provide necessary solutions as well as other materials, such as tubes and columns. Generally, cells are lysed and the DNA is bound in place (for example, to silica on a column), followed by washing and DNA release. The DNA isolated tends to be pure, but sample digestion is short, and thus yield may be sacrificed; yield can particularly suffer when limited amounts of DNA exist.
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Automation (Forensic Science)
The use of robotic means of DNA preparation allows the processing of large numbers of samples in short amounts of time while eliminating the human factor. Automated DNA isolation is most desirable for work with high-quality material, such as database samples. Material involved in law-enforcement investigations is less often processed in this manner. In automated methods, the DNA isolation procedures are similar to those detailed above (particularly those for kits), with reagent transfer being automated. The robots involved are expensive, as are the proprietary reagents required, but the savings in technician time can be substantial.
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Further Reading (Forensic Science)
Belgrader, P., et al. “Automated DNA Purification and Amplification from Blood-Stained Cards Using a Robotic Workstation.” BioTechniques 19 (September, 1995): 426-432. Discusses the automated isolation of DNA from paper matrices.
Butler, John M. Forensic DNA Typing: Biology, Technology, and Genetics of STR Markers. 2d ed. Burlington, Mass.: Elsevier Academic Press, 2005. Provides a detailed overview of the basic biology of DNA and forensic DNA methodologies.
Greenspoon, S. A., et al. “Application of the BioMek 2000 Laboratory Automation Workstation and the DNA IQ System to the Extraction of Forensic Casework Samples.” Journal of Forensic Sciences 49 (2004): 29-39. Reports on the automated isolation of DNA through the use of a commercial kit.
Nagy, M., et al. “Optimization and Validation of a Fully Automated Silica-Coated Magnetic Beads Purification Technology in Forensics.” Forensic Science International 152, no. 1 (2005): 13-22. Describes the automated isolation of DNA using silica.
Walsh, P. S., D. A. Metzger, and R. Higuchi. “Chelex 100 as a Medium for Simple Extraction of DNA for PCR-Based Typing from Forensic Material.” BioTechniques 10 (April, 1991): 506-513. Reports on the first detailed application of the Chelex technique for DNA isolation.
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DNA Isolation Methods (World of Forensic Science)
Deoxyribonucleic acid (DNA) isolation is an extraction process of DNA from various sources. Methods used to isolate DNA are dependent on the source, age, and size of the sample. Despite the wide variety of methods used, there are some similarities among them. In general, they aim to separate DNA present in the nucleus of the cell from other cellular components.
Isolation of DNA is needed for genetic analysis, which is used for scientific, medical, or forensic purposes. Scientists use DNA in a number of applications, such as introduction of DNA into cells and animals or plants, or for diagnostic purposes. In medicine the latter application is the most common. On the other hand, forensic science needs to recover DNA for identification of individuals (for example rapists, petty thieves, accident, or war victims), paternity determination, and plant or animal identification.
Presence of proteins, lipids, polysaccharides and some other organic or inorganic compounds in the DNA preparation can interfere with DNA analysis methods, especially with polymerase chain reaction (PCR). They can also reduce the quality of DNA leading to its shorter storage life.
Sources for DNA isolation are very diverse. Basically it can be isolated from any living or dead organism. Common sources for DNA isolation include whole blood, hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek) swabs, epithelial cells, urine, paper cards used for sample collection, bacteria, animal tissues, or plants.
It is quite clear that the extraction methods have to be adapted in such a way that they can efficiently purify DNA from various sources. Another important factor is the sample size. If the sample is small (for example sperm, or a single hair) the method has to be different to the method used in isolating DNA from a couple of milligrams of tissue or milliliters of blood. Another important factor is whether the sample is fresh or has been stored. Stored samples can come from archived tissue samples, frozen blood or tissue, exhumed bones or tissues, and ancient human, animal, or plant samples.
The isolation of DNA usually begins with lysis, or breakdown, of tissue or cells. This process is essential for the destruction of protein structures and allows for release of nucleic acids from the nucleus. Lysis is carried out in a salt solution, containing detergents to denature proteins or proteases (enzymes digesting proteins), such as Proteinase K, or in some cases both. It results in the breakdown of cells and dissolving of membranes.
While the lysis of soft tissues or cells is easy, DNA also has to be isolated from hard tissues, such as bone, wood, and various plant materials. Most plant samples require freezing in liquid nitrogen and subsequently pulverizing the tissues to a fine powder. On the other hand, bones are highly mineralized and the ions have to be removed from the samples before extraction so they do not later interfere with PCR. Once the samples are partly processed they are then homogenized in lysis buffer using a mechanical homogenizer.
DNA isolation is a simple process and can be performed in a kitchen using household appliances and chemicals. Vegetables or meat can be homogenized with salt and water. After that, by application of a detergent, cellular proteins and lipids are separated away from DNA. Enzymes found in meat tenderizer or pineapple juice allow precipitation of proteins and free DNA into the solution. By adding alcohol to the mix, nucleic acid is brought to the top of the container and can be spooled onto a stick as a visible white string.
A number of commercial DNA purification kits use the very same principles as this household method, but different reagents. In a commercial kit the common lysis solutions contain: sodium chloride; tromethamine (also known as Tris), which is a buffer to retain constant pH; ethylenediaminetetraacetic acid (EDTA), which binds metal ions; and sodium dodecyl sulfate (SDS), which is a detergent. A common enzyme used in DNA extraction is Proteinase K.
The oldest methods of DNA purification in laboratories, still often used also by the FBI, rely on a mix of organic solvents. Lysed samples are mixed with phenol, chloroform, and isoamylalcohol for separation of DNA and protein. Proteins are denatured by the organic mixture. When the sample is centrifuged, DNA is retained in the aqueous (water) layer, phenol is at the bottom of the tube, and denatured proteins form a cloudy interface. This method is very efficient, but unfortunately it can only be used if the quantity of starting material is reasonably abundant. Moreover, the organic solvents used carry health and safety problems. The quality of the DNA from this procedure is usually not adequate for some more sensitive analytical techniques (especially sequencing and occasionally PCR).
A modification of the method uses high salt (sodium chloride, NaCl) concentration to bring down DNA. After the denaturation of cellular proteins using detergents and a protease for a few hours or overnight, salt is added and mixed with the solution. As a result, salt of nucleic acid is formed and in presence of alcohol can be recovered by centrifugation.
Occasionally, alkaline denaturation of the sample is used to release DNA from the cells. Buccal swabs and occasionally blood stains can be placed in small plastic tubes (eppendorfs) and subjected to denaturation with sodium hydroxide (NaOH). The solution is then re-equilibrated to neutral pH with a more acidic buffer solution and is ready for PCR. Although it is a quick and simple method, the quality of DNA is not always adequate for all applications.
A method similar to alkaline denaturation is heat denaturation, achieved by boiling samples. Heating of a sample to 100°C releases DNA into the solution but also denatures it by separating the two strands. In some cases this procedure gives adequate nucleic acid that can be amplified by PCR, however, most of the time there are remaining inhibitors in the form of degraded proteins, other organic compounds, or ions.
A related method used commonly in forensic laboratories utilizes Chelex ion exchange resin that binds multivalent metal ions and is particularly useful in removing inhibitors from DNA. It can be used with any type of sample, including whole blood, bloodstains, seminal stains, buccal swabs, or hair. The only difference from the previous method is the presence of resin, which binds the impurities from the solution, while DNA is being left in the solution. By centrifuging the samples, the resin is brought to a pellet and separated.
Another method similar to Chelex relies on the use of paramagnetic beads with DNA binding capacity. Samples are lysed and then the solid material is treated with Proteinase K. The lysates are then applied to the beads. Resin is subsequently washed and DNA is eluted of it at 65°C, magnetic beads are separated from the sample on a magnetic stand.
Other methods of DNA purification involve columns of various sorts, which are packed with ion exchange, or silica based resins or matrices. Ion exchange columns are generally positively charged to bind the negatively charged DNA; silica matrices are also charged and can also retain DNA. In such applications DNA from the cellular lysates is expected to bind to the column. These columns are then washed using salt solutions to remove unbound material. Nucleic acid is then recovered by applying water or a neutral pH salt solution to break down the resin-DNA bonding.
The use of columns allows increased throughput of samples, shorter time of isolation in comparison to traditional solvent based extraction, increased yield of recovered DNA, and improved quality of purified DNA.
In addition to columns and the previously described resins, there are also liquid resins that are used. The principle is the same as for magnetic beads, but at the final step the samples have to be spun to separate DNA from the resin.
All of these methods so far have dealt with simple, single samples. In some cases a sample consists of a mixture of cells, for example sperm cells and non-sperm epithelial cells. This extraction is based on differential properties of the two cell types. Sperm cells resist Proteinase K lysis; therefore the non-sperm cells are lysed first in its presence. When the tube is centrifuged, the solution contains epithelial DNA, while the pellet contains sperm cells. Sperm cells are subsequently lysed by adding dithiothreitol or DTT with Proteinase K. Any of the techniques mentioned before can be used to isolate the DNA from those differential lysates.
Although plants are not a common source of DNA for forensic investigation, analysis of their DNA is very common in science. Plants are more difficult to work with than many other materials for a couple of reasons. First, plant cells have a cell wall, which has to be at least partly destroyed before the cytoplasm with the DNA can be accessed. Second, plants often have high levels of sugars (for example starch or fructose) in their tissues or other organic compounds such as polyphenols.
Grinding of the samples in liquid nitrogen helps to destroy the cell wall, but the organic compounds including sugars still remain. As a result, methods were developed that use chloroform-octanol mix, hexadecyltrimethylammonium bromide (CTAB) with high salt to remove polysaccharides, and polyvinylpyrrolidone (PVP) to remove polyphenols.
All of these methods are successfully used in various laboratories and with various samples. The methods have to be properly selected to optimize the yield and quality of the DNA extracted.
SEE ALSO DNA; DNA profiling; DNA typing systems.