DNA isolation methods

DEFINITION: Techniques used to obtain DNA specimens from biological materials.

SIGNIFICANCE: DNA obtained from forensic samples can be used to link suspects to crime scenes, associate suspects and victims, identify the remains of missing individuals, or determine parentage. Numerous techniques are available for isolating DNA from cellular material; the choice of the most appropriate helps to ensure that a DNA profile will be obtained successfully.

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. These all involve breaking open cells (lysis) to release DNA, purification to remove unwanted material, and harvesting the DNA for analysis. Methods developed in the twenty-first century included magnetic and silica-based extraction.

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 be worn, and equipment must be cleaned regularly. A reagent blank control, containing no tissue but undergoing the same extraction process, should be included to ensure that reagents are not contaminated.

Organic Extraction

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.

Differential Extraction

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.

Chelex Extraction

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.

Preservative Papers

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.

Commercial DNA Isolation Kits

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.

Automation

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.

Bibliography

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