A simple method for extraction of DNA from fungi and yeasts with anhydrous hydrogen fluoride

Oshiro, Satoru; Katsura, Nobuhiko; Kitada, Koji; Gunge, Norio

doi:10.1016/0014-5793(87)80851-7

Cited by 6 publications

References 11 publications

(17 reference statements)

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DNAExtraction Methods in Forensic Analysis

Cattaneo

Gelsthorpe²,

Sokol³

2000

Encyclopedia of Analytical Chemistry

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Advances in molecular biology can be applied to forensic practice with spectacular success. However, the extraction of DNA from scarce or badly degraded substances remains a significant problem and an ability to overcome it remains crucial because the success of all DNA analyses depend on a successful initial extraction. Extraction methods must solve problems associated with low quantities of DNA, DNA degradation, polymerase chain reaction (PCR) inhibitors and failure to solubilize or separate DNA from the substrate. These problems are compounded by the relative lack of knowledge concerning DNA degradation in different forensic environments. The extraction method must be suitable for the starting material. Fresh material with abundant DNA, such as blood, soft tissue, or saliva, presents no difficulties and DNA suitable for PCR analysis can be extracted easily. In mixed substrates, for instance vaginal swabs from rape victims, male and female DNA can be separated by differential lysis procedures. Putrefied material with abundant DNA, such as blood, soft tissues, and entomological and botanical material, has plenty of DNA, but PCR inhibitors may be present and extraction methods must be chosen that select DNA but not the inhibitors. Clean material with scarce DNA, such as stains (blood, saliva, sperm), swabs (vaginal and from bitemarks, fingernails and stains on bodies), fingernails, hair, desquamated cells (skin, dandruff), and certain body liquids (urine, plasma), has a paucity of cellular material and hence low quantities of DNA. The prerequisite here is to reduce the loss of DNA, for example by introducing more specific methods of extraction and reducing the number of steps in the procedure. In the case of dirty material with scarce DNA, such as sperm, saliva and blood stains, nails and feces, not only are there low quantities of DNA, but there are also large amounts of organic and inorganic contamination. The problems faced by any extraction method are to recuperate small quantities of DNA as well as eliminating inhibitors of PCR. Formalin‐fixed and paraffin‐embedded tissues and stained smears on slides present particular problems associated with damage to DNA due to the processing of the materials. Nevertheless, DNA can be successfully extracted from these substrates. The greatest challenge in forensic DNA analysis is extraction from calcified tissues (bone and teeth). These substrates have obviously undergone considerable degradation and are often contaminated with soil. In addition, DNA undergoes binding interactions with the hydroxyapatite matrix which chemically stabilizes DNA; this aids its survival, but makes it difficult to extract. An essential part of the extraction process is therefore to release DNA from this binding. Before DNA can be isolated from a substrate, several important steps must be taken. These involve lysing the cells to release the DNA and digesting protein so that as much DNA as possible is solubilized. Many (if not most) techniques – and there are many variations – involve the use of proteinase K (PK) to digest protein and a detergent (often sodium dodecyl sulfate (SDS)) to lyse the cell membranes. The extraction procedures themselves can be divided into two categories: nonaffinity methods where all extraneous material is removed leaving DNA in solution, and affinity methods whereby the DNA is targeted directly and removed from the solution. Each category has many variations and on occasions they can be combined. The main nonaffinity techniques are phenol/chloroform, salt precipitation and chelex. The phenol/chloroform method is the best known and has an excellent forensic record; it is based on removing protein, thus purifying the nucleic acids which are extracted in aqueous solution; the disadvantages are that it does not remove nonprotein contaminants, several steps are involved in the procedure, and the reagents are toxic. The salt precipitation method is based on salting‐out by dehydration and precipitation with a saturated salt solution. It is an excellent method when abundant clean samples are available and is quicker, cheaper and less toxic than the phenol/chloroform procedure. The disadvantages are that it does not eliminate all PCR inhibitors and considerable amounts of protein may be retained. With chelation (e.g. chelex), the active reagent binds to inorganic substances and clears the extract of certain PCR inhibitors, particularly metal ions; an alkaline pH disrupts cell membranes leaving DNA in solution. The method is simple, rapid and does not involve multiple tube transfers, though it does not have a particularly selective action on protein. The novelty of affinity methods is that they actively select DNA by either specific or nonspecific reactions. The main nonspecific affinity method is the glass–milk or silica gel procedure. Very small glass beads are combined with isothiocyanate which itself is bound to guanidium. The positively charged amine groups on the guanidium link with the negatively charged phosphate groups on DNA. Once bound to the glass beads, the DNA can be repeatedly washed and cleansed of all other material before being eluted and subjected to PCR analysis. This is currently the method of choice for difficult substrates; the disadvantage is that when a great deal of contaminant is present, it might sterically hinder the adsorption process of DNA on to the glass substrate. The only specific affinity method yet published uses a murine anti‐dsDNA monoclonal antibody of IgG2A subclass bound to paramagnetic beads via anti‐murine IgG. The antibody‐coated beads react specifically with DNA and can easily be washed free of contaminants. The method is extremely specific and sensitive, positive PCR results being obtainable from as little as 0.005 ng per microliter of DNA. In addition, the bead–anti‐DNA–DNA complex can be put directly into the PCR. As yet this method has not been fully tested on all forensic substrates, but initial results are very promising. Commercial DNA extraction kits, for example Dynal DNA direct™, QIAamp ® , Prep‐A‐Gene ® and Scotlab Geneclean ® , are becoming increasingly available and some provide protocols for forensic substrates. Most of these kits are based on an affinity mechanism. In practice they seem to give reliable results. In conclusion, DNA extraction methods in forensic analysis seem to be going two ways. For substrates with fresh abundant DNA, extraction presents no problems and any method would suffice; it would seem logical to choose one that uses nontoxic reagents and is inexpensive. The most interesting evolution in extraction techniques is with those dealing with difficult substrates where DNA is scarce or tightly bound and non‐DNA contamination is abundant. Recent work suggests that the future in these situations lies with affinity methods and the development of immunoaffinity seems to point the way for future research. Such studies should be combined with investigations into the interaction between DNA and forensic environments so that extraction procedures can be devised for particular circumstances, for only with proper extraction procedures will subsequent DNA analysis be successful.

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