An alternative procedure for DNA extraction that has become popular among forensic scientists is the use of a chelating-resin suspension that can be added directly to the sample (e.g., blood, bloodstain, or semen). Introduced in 1991 to the forensic DNA community, Chelex® 100 (Bio-Rad Laboratories, Hercules, CA) is an ion-exchange resin that is added as a suspension to the samples (Walsh et al. 1991). Chelex is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions that act as chelating groups in binding polyvalent metal ions such as magnesium. Like iron filings to a magnet, the magnesium ions are drawn in and bound up. By removing the magnesium from the reaction, DNA destroying enzymes known as nucleases are inactivated and the DNA molecules are thus protected.
In most protocols, biological samples such as bloodstains are added to a 5% Chelex suspension and boiled for several minutes to break open the cells and release the DNA. An initial, prior wash step is helpful to remove possible contaminants and inhibitors such as heme and other proteins (Willard et al. 1998). The exposure to 100°C temperatures denatures the DNA as well as disrupting the cell membranes and destroying the cell proteins. After a quick spin in a centrifuge to pull the Chelex resin and cellular debris to the bottom of the tube, the supernatant is removed and can be added directly to the PCR amplification reaction.
Chelex extraction procedures for recovering DNA from bloodstains or semen-containing stains are not effective for RFLP typing because Chelex denatures double-stranded DNA and yields single-stranded DNA from the extraction process.
Thus, it can only be followed by PCR-based analyses (see Table 2.1). However, Chelex extraction is an advantage for PCR-based typing methods because it removes inhibitors of PCR and uses only a single tube for the DNA extraction, which reduces the potential for laboratory-induced contamination.
The addition of too much whole blood or too large a bloodstain to the Chelex extraction solution can result in some PCR inhibition. The AmpFlSTR kit manuals recommend 3 ||L whole blood or a bloodstain approximately 3 mm X 3 mm (Applied Biosystems 1998).
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