Procedures utilizing Chelex100 chelating resin have been developed for extracting DNA from forensic-type samples for use with the PCR. The procedures are simple, rapid, involve no organic solvents and do not require multiple tube transfers for most types of samples. The extracion of DNA from semen and very small blood stains using Chelex 100 is as efficient or more efficient than using proteinase K and phenol-chloroform extraction. DNA extracted from bloodstains seems less prone to contain PCR inhibitors when prepared by this method. The Chelex method has been used with amplification and typing at the HLA DQα locus to obtain the DQα genotypes of many different types of samples, including whole blood, bloodstains, seminal stains, buccal swabs, hair and post-coital samples. The results of a concordance study are presented in which the DQα genotypes of 84 samples prepared using Chelex or using conventional phenol-chloroform extraction are compared. The genotypes obtained using the two different extraction methods were identical for all samples tested.
Methods of extracting DNA from a variety of biological materials have historically included separation and purification steps utilizing phenolchloroform extractions and/or ethanol precipitation (3, 8). Inorganic extraction procedures have included the use of high salt concentration (4), excess proteinase K digestion (5) and the use of glass powder (13). Although these methods are successful in recovering high molecular weight DNA from large samples, they require several steps and may include transfers of DNA extracts to additional containers or washing/desalting procedures using various commercial filters or columns. These additional steps allow increased opportunities for cross-transfer of samples or introduction of contaminants.
The polymerase chain reaction (PCR) (12) is a method of amplifying small quantities of relatively short target sequences of DNA using sequencespecific oligonucleotide primers and thermostable Taq DNA polymerase. PCR does not require native high molecular weight DNA in order to amplify target sequences. Only the target sequence itself needs to be intact. Thus, PCR can amplify partially degraded and/or denatured DNA. PCR has great potential benefit for the analysis of forensic casework samples, in which the amount and molecular weight of DNA may be less than optimal for analysis by other methods.
Recently, Singer-Sam et al. reported the use of Chelex 100 as a means of increasing the signal from the PCR amplification of small amounts of DNA released from small numbers of tissue culture cells that have been boiled (11). Chelex I00 is a chelating resin that has a high affinity for polyvalent metal ions. The Chelex resin is composed of styrene divinylbenzene copolymers containing paired iminodiacetate ions, which act as chelating groups. Singer-Sam et al. postulated that the presence of Chelex during boiling prevents the degradation of DNA by chelating metal ions that may act as catalysts in the breakdown of DNA at high temperatures in low ionic strength solutions (11).
The procedures adapted for forensic samples are based on those used by Singer- Sam et al. and involve boiling suspensions of cells in a 5% suspension of Chelex, followed by amplification of a fraction of the supernatant by PCR. The alkalinity of Chelex suspensions (pH 10-11)and the exposure to 100°C temperatures result in disruption of the cell membranes and denaturation of the DNA. Because these Chelex procedures result in denatured sample DNA, the DNA is not suitable for RFLP analysis.
In this paper, we demonstrate that the Chelex extraction method efficiently makes available to PCR amplification DNA from sperm and from blood dried onto cloth. Also, we show that for all 84 different forensic-type samples examined, there are no differences between the HLA DQα genotypes obtained by PCR amplification of DNA prepared by either conventional phenol-chloroform extraction or the Chelex-based procedure.Materials and methods Assessment of Chelex DNA extraction from semen
Fresh semen, which had been stored frozen, was thawed and its sperm content measured using a hemocytometer. Five microliters of this semen were added in duplicate either to 200 µl of 5% Chelex 100 (Bio-Rad, Richmond, CA) or to 200 µl of sterile distilled water. Both Chelex-and water-treated samples were carried through the remainder of the extraction protocol described below for semen. To estimate DNA released, 4’,6’-diamidino-2- phenylindole (DAPI)fluorescence measurements were taken on the resultant supernatant (2, 7). DNA in amounts estimated by DAPI fluorescence and input sperm count were added to HLA-DQα PCR as described below, and the yield of PCR product was compared by gel electrophoresis (1, 8) to that obtained from comparable amounts of highly-purified control DNA extracted with phenol-chloroform.Assessment of Chelex DNA extraction from bloodstains
Fresh blood from a fingerprick was spotted in duplicate onto cotton cloth in 5-,2-, and 1-µl volumes. The same volumes of blood were also spotted onto Saran Wrap (Dow Brands, Indianapolis, IN). The stains were kept in the dark for 1 week. The stains on cotton cloth were either treated as described below for DNA extraction using Chelex or by organic extraction (1, 8). The dried blood on Saran Wrap was resuspended in 50 µl of sterile distilled water, and 10- or 3-µl aliquots of these resuspensions were extracted either by the Chelex procedure or by the use of organic solvent. Ten percent of the total extract of a stain by either method was added to an HLA-DQα PCR, as described.