Gel electrophoresis of amplification products was performed using 3 NuSieve/1%GTG agarose gels (FMC, BioProducts, Rockland, ME). Both the gel buffer and running buffer were 89 mM Tris-borate, 2.5 mM EDTA pH 8.3 (1).Results Chelex DNA extraction from semen
Aliquots of DNA prepared from semen by Chelex extraction as described above were amplified for HLAeach aliquot was estimated by DAPI fluorometry and the input number of sperm cells. For DNA prepared using either Chelex or using water without Chelex, the fluorescence measurement of DNA was about 45%-80% of that predicted by the number of sperm. The yield of PCR product DNA was revealed by gel electrophoresis as shown in Figure 1. The input amount of DNA estimated by fluorometry is given, as are the amounts of input highly-purified control DNA. The yield of PCR product from Chelex-preparcd DNA is comparable to that from purified DNA, while DNA prepared without Chelex results in much less amplified DNA.
Chelex DNA extraction from bloodstains
Small bloodstains had been prepared as described above. Stains were prepared either on cloth or on plastic wrap. The stains on plastic were done to allow complete resuspension and aliquoting of fractions of the resuspension in order to quantify very small volumes of blood that had dried. Extracts of various size stains were prepared either by using Chelex or phenol-chloroform extractions. Ten percent of each extract was subjected to PCR amplification for the DQα locus. The results of the amplification as assayed by gel electrophoresis are shown in Figure 2. In general, there is little difference in PCR product yield from DNA prepared by either method. When there is a difference, the yield from Chelex-prepared DNA is greater.
Forensic-type samples: concordance study
The different forensic-type samples listed in Tables 1234 (a total of 84 samples) have all been previously typed at the DQα locus after their DNA had been extracted using traditional phenol-chloroform methodology (1, 8). After repeating the extraction of another portion or replicate of each of these samples, but using the Chelex-based procedures described above, the typings were repeated. As summarized in Table 5, there were no discrepencies for any samples in the DQα genotype obtained using either extraction method. Representative probe strip typings of 16 of these samples are shown in Figure 3.
Among these samples. one bloodstain (#107, Table 3) and one semen stain (#133, Table 3) failed to amplify initially using the Chelex procedure. The semen stain was suocessfully amplified and typed after repeating the extraction from a fresh cutting from the fabric substrate. The bloodstain substrate was a red fabric which colored the extract a red color. This red dye apparently inhibited the amplification.
An additional 3 x 3-mm piece of this bloodstain was allowed to soak for 30 min at room temperature, and then it was subjected to vortexing to dislodge cellular material from the fabric. The fabric was then removed and the solution centrifuged to pellet the cellular debris. The supernatant was removed and discarded. The cellular debris was washed three times with water washes, followed by centrifugation. All but 50ml of the last wash was discarded, and 150ml of 5% Chelex was added. The remainder of the protocol was as described above. At this time the sample was successfully amplified and typed.Typing from single, stained threads
The single 3-mm stained yarns cut from 2 different bloodstains and one semen stain were successfully typed.Discussion
A set of protocols have been described here which effectively and simply extract DNA from a variety of materials, including those encountered in forensic science laboratories. The extracted DNA can be amplified by PCR and hybridized to allele-specific oligonucleotide probes. Because of the relative simplicity of these procedures, it is anticipated that this will make the use of PCR in forensic analyses more easily accomplished. The reduction of the number of steps in sample preparation in which sample is transferred will also help reduce the chance of operator-introduced DNA contamination or of sample mix-up.