This study performed the semen discrimination test, short tandem repeat (STR) typing, and Y-chromosome specific-STR (Y-STR) typing on five, 30–50-year-old semen stains. All samples reacted positively with the SM test reagent, and we observed sperm heads in all samples microscopically. The quantity of DNA extracted from the 43- and 50-year-old samples was much lower than from the other samples. STR typing of the 30-, 32-, 32-, 43-, and 50-year-old semen samples detected a maximum of 13, 15, 15, 11, and 6 of 15 loci, respectively, while Y-STR typing detected 16, 16, 16, 10, and 10 of 16 loci. These results suggest that the semen discrimination test and STR and Y-STR typing can detect extremely old semen stains and are useful for forensic practice.
Semen stains left at the scene constitute important evidence in sex crimes because the identification of semen is useful for understanding the details of the crime and because DNA typing can identify a suspect. The results of short tandem repeat (STR) typing of very old semen stains have become important evidence in long-unsolved sex crimes. However, the usefulness of various forensic examinations; i.e., semen discrimination and DNA typing, has not been evaluated for extremely old semen stains. If these examinations were useful for such extremely old stains, they would help to determine the outcomes of sex cases. Therefore, we evaluated whether STR and Y-chromosome specific-STR (Y-STR) typing of semen stains more than 30 years old provides useful forensic evidence.
2. Materials and methods
We examined five semen stains stored at room temperature for 30, 32, 32, 43, and 50 years. This study was approved by the Ethics Committee of Saitama Medical University (No. 673).
2.2 Discrimination test for semen
The acid phosphatase assay and microscopy visualization were performed as discriminatory tests for semen. For the preliminary discrimination of semen, the acid phosphatase test was performed using SM-test reagent (Wako, Osaka, Japan), according to the manufacturer's protocol. SM-test reagent was dropped on a piece of gauze directly. The sample stained with SM-test reagent was incubated at room temperature. We defined a positive result as purple coloration visible by the naked eye after a 5-min incubation.
Sperm heads were confirmed microscopically by Baecchi staining. Sperm in a 0.5-cm × 0.5-cm piece of gauze was removed with 20 μL of distilled water. The resulting solution was immobilized on a glass slide, and then subjected to Baecchi staining.
2.3 DNA extraction and human-DNA quantification
DNA was extracted and purified with an EZ1 Investigator Kit (QIAGEN, CA, USA), a QIAamp DNA Mini Kit (QIAGEN), ISOHAIR (NIPPON GENE, Tokyo, Japan), and a DNA Extractor FM kit (Wako) using a 1-cm × 0.5-cm piece of gauze, according to the manufacturers’ protocols, and then eluted using 50 μL of water.
The quantity and purity of the DNA were evaluated by measuring the OD260 and OD260/280 spectrophotometrically (NanoDrop 1000; Thermo Fisher Scientific, Waltham, MA, USA). Human DNA, in the extracts was quantified using a Human-DNA Quantification Kit (Takara, Shiga, Japan), which amplifies a 207-bp fragment of the D17Z1 region, according to the manufacturer's protocol.
2.4 STR and Y-STR typing
Single tandem repeat typing was performed using the AmpFlSTR® Identifiler™ and Yfiler™ PCR amplification kits (Life Technologies, Carlsbad, CA, USA), according to the manufacturer's protocol. STR alleles were detected on an ABI 310 Genetic Analyzer (Life Technologies), following analysis using the GeneMapper™ ID software (Life Technologies). The positive threshold of peak height was set at greater than 150 relative fluorescent units (RFUs).
3. Results and discussion
All samples reacted positively with the SM test reagent and we observed sperm heads in all samples microscopically. This suggested that even extremely old stains could be identified as semen.
On quantifying the DNA using the OD260, all samples had sufficient DNA for STR typing, although on quantifying the DNA using real-time PCR, much less DNA was extracted from the 43- and 50-year-old samples, and there was insufficient DNA for STR typing (Table 1). This suggests that the DNA extracted from the older two samples had been degraded so that the fragments were smaller than 207 bp. With extremely old semen stains, the degree of degradation of the extracted DNA might affect STR typing, so different DNA quantification methods might be required. Of the four commercial kits, the DNA Extractor FM kit extracted the greatest quantity of DNA.
Table 1The concentration of DNA extracted using various commercial kits measured using the OD260 and real time PCR.
|Storage duration (years)||DNA concentration by OD260 (ng/ml)||DNA concentration by real-time PCR (ng/ml)|
Fig. 1 shows the number of loci detected in each sample and the electropherograms resulting from STR and Y-STR typing. If even one peak appeared, the number of loci detected was determined. Using the AmpFlSTR® Identifiler™ kit, STR typing of the 30-, 32-, 32-, 43-, and 50-year-old semen samples detected a maximum of 13, 15, 15, 11, and 6 of 15 loci, respectively. For the 43- and 50-year-old semen samples, the longest fragments detected were of 269 and 208 bp, respectively. In comparison, using the AmpFlSTR® Yfiler™ kit, Y-STR typing detected a maximum of 16, 16, 16, 10, and 10 of 16 loci, respectively. Hara et al. reported that STR and Y-STR typing could be used on 25-year-old semen stains [
]. However, our results indicate that STR and Y-STR typing can be used on stains older than 30 years.
- Hara M.
- Kido A.
- Takada A.
- et al.
STR and Y-STR genotyping assays of 25-year-old semen stains.
Forensic Sci. Int. Genet. Suppl. 2008; 1: 428-429
In conclusion, the semen discrimination test and STR and Y-STR typing could be used to obtain forensic evidence from extremely old semen stains.
- STR and Y-STR genotyping assays of 25-year-old semen stains.Forensic Sci. Int. Genet. Suppl. 2008; 1: 428-429
Published online: October 28, 2013
Accepted: October 2, 2013
Received: August 28, 2013
© 2013 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.