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Figure 3.1 Principles of extraction techniques commonly used in forensic DNA laboratories. Part (c) reproduced with permission from QIAGEN Products phase and discarded. The aqueous supernatant is transferred to a new tube and an equilibrated mixture of phenol-chloroform-isoamyl alcohol in the ratio of 25 : 24 : 1 is added to ensure complete removal of the proteins. After agitation, the mixture is centrifuged and the resultant aqueous supernatant is transferred again to a new tube and a chloroform-isoamyl alcohol mixture in the ratio of 24 :1 is added and followed by further mixing and centrifugation in order to ensure the complete removal of phenol. The aqueous supernatant is collected, and an acetate salt and 2 volumes of 96% ice-cold ethanol are added to precipitate the DNA via shielding of the negative charges on DNA, allowing it to aggregate and precipitate. Samples are incubated at -20°C to increase the efficiency of precipitation, and are then centrifuged to collect the precipitated DNA. Ethanol is decanted and the DNA pellet is washed with 70% ethanol to remove the salt, then vacuum- or air-dried and resuspended in either sterile double-distilled water or low-salt buffer. Some protocols involve an additional purification and concentration step with spin columns, which remove inhibitors with solubility characteristics similar to DNA, such as heme (Akane et al., 1994) or indigo dyes.

Phenol-chloroform extraction works very well for the recovery of double-stranded high-molecular-weight DNA. However, the method is time-consuming, involves the use of hazardous chemicals and requires multiple tube transfers as well as a final precipitation step, potentially increasing the risk of contamination and/or sample mix-ups. Nevertheless, the method works very well for extraction of DNA from nearly all of the common types of forensic samples, and is still used today as a last resort for DNA extraction from problematic samples because it produces relatively large yields of high-quality DNA, and the excellent DNA purity allows it to remain stable in long storage. In this respect, phenolchloroform extraction remains the gold standard by which new methods are judged.

Chelex® 100 extraction

Chelex® 100 is a medium used for the simple extraction of DNA (Figure 3.1b) for subsequent use in PCR-based typing. It is a styrene divinylbenzene copoly-mer containing paired iminodiacetate ions, which act as chelating groups in the binding of polyvalent metal ions such as magnesium or calcium (Instruction Manual, Chelex® 100, Bio-Rad Laboratories, Hercules, CA). Chelex therefore binds bivalent ions such as Ca2+ and Mg2+ and deactivates unwanted enzymes such as DNases. Chelation of these cations may also result in the 'deactivation', via structural changes, of proteins that make up the cellular architecture, leading to the destabilization of the whole cell and essentially resulting in cellular lysis. Addition of proteinase K, a Ca2+-independent enzyme, breaks down the deactivated enzymes and proteins. Proteinase K is then itself deactivated by a subsequent boiling step. This method was introduced into forensic laboratories on the basis of a protocol by Walsh et al. (1991): biological samples are added to a 5% Chelex® 100 resin, are boiled for several minutes and are then centrifuged to remove the Chelex resin, leaving the DNA in the supernatant. The single-stranded DNA can be used directly in PCR. Better results are often obtained if an initial incubation step is used in order to remove the 'forensic stain' from the carrier. Besides, several protocols call for the additional incubation of 100 ng of proteinase K with the Chelex/stain mixture for two hours at 56°C or overnight at 37°C. The Chelex® 100-based extraction has a definite advantage over other methods in that it is very fast and can also be carried out in a single tube without any transfer steps, which substantially reduces the possibility of contamination. However, DNA extracted by Chelex® 100 requires further purification to reach the level of DNA quality obtained from a standard phenol-chloroform extraction.

Magnetic affinity solid-phase extraction

Solid-phase DNA extractions have been in use for many years. In the advanced case of magnetic affinity solid-phase extraction, efficient DNA isolation relies on the binding of DNA to a silica surface on paramagnetic beads in the presence of chaotropic solutions (Figure 3.1c). DNA isolation can therefore be performed in a single tube by adding and removing solutions such that contaminants are washed away while the DNA remains bound to the beads (until its eventual elution). Solid-phase extraction approaches are marketed by Qiagen GmbH, Hilden, Germany (MagAttract® DNA chemistry) and Promega Corporation, Madison, USA (DNA IQTM system). This extraction method is made possible by the tendency of DNA to bind to silica (glass) in the presence of chaotropic salts such as sodium iodide, guanidinium thiocyanate (GTC) or guanidinium hydrochloride. Cells are first lysed in a lysis and binding solution so that DNA is released. DNases are denatured and inactivated by the presence of the chao-tropic salts, and magnetic beads are then mixed with the sample to allow DNA to bind. DNA requires high salt conditions to bind to the silica surface on the beads, but this DNA binding is reversible at pH <7.5, so when the washing steps are complete the DNA may be eluted by water or by a low-salt buffer. After binding, the magnetic beads containing the immobilized DNA are collected by simply applying a magnetic force. The soluble portion containing the unbound components (proteins, cell debris' etc.) is then removed and discarded. The magnetic particles with the attached DNA are then resuspended in a series of wash solutions in order to obtain highly purified DNA: a solution of chaotropic salts removes residual non-bound matter, ethanol removes residual chaotropic salts and a short rinse with water removes ethanol. The DNA is finally eluted from the magnetic beads by the addition of either water or low-salt buffer and is ready for use in downstream applications. Magnetic bead-based DNA isolation has several advantages over both the phenol-chloroform and Chelex® 100 methods, including:

• elimination of traditional solvent extraction, thus avoiding the use of harmful organic solvents,

• elimination of precipitation, centrifugation and pellet-drying steps,

• rapid purification,

• removal of nearly all contaminants that could interfere with subsequent PCR,

• production of high-quality single-stranded DNA,

• scalable and reproducible extraction,

• suitability for adaptation for high-throughput extraction.

3.4 Modified techniques for DNA extraction from challenging forensic samples

Magnetic bead-based purification is currently the technique best suited for DNA extraction from the majority of forensic samples. However, some sample types, such as sperm and skeletal remains, pose special challenges. For these more recalcitrant samples, extraction techniques must be modified in order to successfully extract usable DNA, as we discuss in the following sections.

Sperm extraction - differential extraction

A special differential lysis treatment for forensic samples from sexual assault cases can separate epithelial cells from sperm cells; the most commonly used method was first described by Gill et al. (1985). Sperm nuclei are resistant to lysis by the usual SDS/proteinase K cell method, but can be lysed in a solution containing proteinase K plus the reductant dithiothreitol (DTT, e.g. 20 |l of 0.1 m DTT is added to 500 |l of lysis buffer), which breaks down the protein disulphide bridges present in sperm nuclear membranes. This method, known as differential lysis, is often used in forensic laboratories to separate sperm nuclei from vaginal cellular debris in samples obtained from semen-contaminated vaginal swabs, thus enabling the identification of cells specific to the male suspect in a sample of predominantly female cells. However, sperm cells would be absent in a number of situations: some perpetrators of sexual assaults could have a vasectomy or could be azoospermic (lacking any viable sperm), or may have a condition of either retrograde ejaculation (an emission of semen back into the bladder) or anejaculation (complete failure to emit semen). The code and spirit of criminal law states that a violation exists when a sexual act is enforced that highly humiliates the victim, especially when this act entails forced

Figure 3.2 Scheme of Selective extraction versus Y-chromosomal analysis: (a) extraction of male and female epithelial cells after cell lysis and detection of autosomal STR profiles; (b) differential extraction to separate male sperm cells from female epithelial cells and detection of autosomal STR profiles; (c) extraction of male and female epithelial cells as well as male sperm cells after cell lysis with DTT and direct analysis of the female (autosomal) and male (Y) STR profile

Figure 3.2 Scheme of Selective extraction versus Y-chromosomal analysis: (a) extraction of male and female epithelial cells after cell lysis and detection of autosomal STR profiles; (b) differential extraction to separate male sperm cells from female epithelial cells and detection of autosomal STR profiles; (c) extraction of male and female epithelial cells as well as male sperm cells after cell lysis with DTT and direct analysis of the female (autosomal) and male (Y) STR profile penetration into the body (§177 StGB, Germany: Gesetzestext mit Rechtssprechung via dejure.org). In the special cases described above, a forensic examination would not reveal sperm cells but would reveal male-specific epithelial cells, providing evidence of a violation. Male DNA profiles can also be picked out of a background of predominantly female cells by analysing for the presence of Y chromosome-specific markers. This Y-specific short tandem repeat (STR) hap-lotype analysis, which is now as sensitive as the autosomal analysis, has superseded and replaced differential extraction in many forensic laboratories (Prinz et al., 2001; Parson et al., 2003; Nagy et al., 2005). As an indication for sperm cells, DTT is generally added to the cell lysis buffer. Figure 3.2 illustrates the principles behind both methods.

Extraction of DNA from bone

There are several protocols that describe different methods for the extraction of DNA from bones. All methods include freezing of the bone in liquid nitrogen and pulverization with a laboratory mill or a dentist's drill, often followed by decalcification to remove the bone matrix. Such methods were applied in the high-throughput bone-fragment-based DNA identification that was necessary after mass disasters such as the World Trade Center attack (Holland et al., 2003) or the Southeastern Asia tsunami disaster (Steinlechner et al., 2005). The methods differ from each other by what is used for the decalcification step. Prado et al. (1997) used only a short decalcification step that entailed overnight incubation of the bone with 0.5 m EDTA, while Holland et al. (1993) repeated this EDTA step three times. Hoss and Paabo (1993) extracted the DNA from bone meal without a decalcification step, and instead used guanidinium thiocyanate and the detergent Triton X-100 for cell lysis. However, in this case DNA could be extracted only from those cells that were exposed after bone pulverization. In a compact bone structure, the density of the surrounding cells provides protection against bacterial degradation for a time, but the density also makes it necessary to provide a thorough decalcification step to release the cells from the matrix (Holland et al., 1993). This decalcification step was noted to be especially important in cases of DNA extraction from older bones (Nagy et al., 2005).

However, the need for the initial steps of manual extraction (freezing, grinding) in all of these methods makes it difficult to apply automation to the extraction of DNA from bone. When automation is required as part of a high-throughput effort, the only means by which it can be incorporated into part of the procedure is as follows: manual pre-extraction treatment is followed by thorough decalcification with EDTA. Bulk material is separated by precise centrifugation, and cellular material is lysed, after which a sensitive automated DNA extraction procedure may be employed.

3.5 Automation of DNA extraction

Automated procedures for forensic DNA analyses are a key for high-throughput sample preparation as well as for avoidance of errors during routine sample preparation and for reproducible processing and improved sample tracking. The most important stage in PCR-based forensic analysis is DNA isolation, and both high yields and high purity are vital components of a successful analysis. A wide variety of high-quality automated instruments, each with its own unique benefits and features, are currently available. An overview of the various instruments and the isolation chemistry that each uses is given in Table 3.1. The National Genetics Reference Laboratory (Mattocks, November 2004, Evaluation Report, NGRL, Wessex, UK: http://www.ngrl.co.uk/Wessex/extraction.htm) evaluated eight automated extraction systems covering both dedicated instruments with their associated chemistries (so-called 'integrated systems') as well as kit-based chemistries that may be suitable for automation when adapted to standard liquid-handling robotic systems. These kits cover several different types of chemistry, including salt extraction, filter plate-based solid-phase extraction and

Table 3.1 Overview of different automated DNA extraction systems

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