Short CommunicationRelationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples
Introduction
If crimes remain unsolved for many years, body fluid and blood stains can be re-investigated using DNA typing methods such as short tandem repeat (STR) typing. Using mock forensic samples, Nakanishi et al. reported that STR loci could be detected (using a commercial kit) from seminal stains stored at room temperature for more than 30 years [1]. However, evidence collected from real crime scenes will have been exposed to a variety of environmental conditions, and the extent of DNA degradation will be unknown. Moreover, because the amounts of DNA extracted from evidentiary samples are often very low, STR evaluation may be of limited value. To identify an individual effectively, it is important to estimate how many loci may be detected using an STR kit.
DNA degradation can be estimated by quantifying DNA fragments of various lengths via quantitative PCR [2], [3], [4]. Briefly, the ratio of short to long amplicons provides an indication of the extent of DNA degradation. However, studies on the relationships between DNA degradation ratios and the number of loci detectable by STR kits are very rare. In the only relevant report, Kitayama et al. suggested that the number of loci detected via STR analysis could be estimated using real-time PCR to calculate the ratios of the amounts of DNA fragments 207 and 98 bp in length in bloodstains containing degraded DNA [5]. However, given that extremely degraded DNA may include fragments below 98 bp in length, the optimal denominator of the degradation ratio may be less than 98 bp.
In the present study, we used a commercial kit (that can quantify 41 bp fragments) to examine extremely degraded DNA. Next, using extremely old seminal stains, we evaluated the relationships between DNA degradation ratios and the numbers of loci detected by three STR kits. Finally, we investigated the relationships between DNA degradation ratios and storage duration.
Section snippets
Samples
We analyzed 19 seminal stains that had been stored at room temperature (11 for 32–39 years, 2 for 40–49 years, 5 for 50–56 years, and 1 for more than 60 years). All stains yielded positive reactions for prostatic acid phosphatase. In addition, sperm heads were evident in all stains using the Baecchi staining method followed by microscopy. The Ethics Committee of Saitama Medical University approved the study protocol.
DNA extraction and quantification
DNA was extracted and purified from 3 to 10 mg samples of sperm-stained gauze using
Relationships between DNA degradation ratios and storage duration
The storage durations and DNA degradation ratios for all 19 samples are shown in Table 1. The 129:41 ratios could be calculated for all samples, but the 305:41 ratios could not be calculated for 6 of the 19 samples because no 305 bp fragments were detected. The relationships between DNA degradation ratios and storage duration are shown in Fig. 1. Both the 129:41 and 305:41 ratios were moderately correlated with storage duration (129:41; r = −0.698, 305:41; r = −0.550). However, if the storage
Conclusion
Storage duration cannot be accurately estimated using DNA degradation ratios, as the ratios were not strongly correlated with duration. However, DNA degradation ratios may indicate whether the storage duration is more than 40 years. The number of loci detected using STR kits correlated with the DNA degradation ratios, especially the 129:41 ratio. Therefore, if in practice a sample is expected to be degraded, the number of detectable STR loci may be estimated by measuring DNA degradation ratio.
Acknowledgments
The authors thank Dr. Takeshi Ohmori (National Research Institute of Police Science) for proofreading this article.
References (8)
- et al.
Evaluation of forensic examination of extremely aged seminal stains
Legal Med
(2014) - et al.
Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies
Forensic Sci Int
(2004) - et al.
A quantitative PCR assay for the assessment of DNA degradation in forensic samples
Forensic Sci Int
(2006) - et al.
Quantification of damage in DNA recovered from highly degraded samples–a case study on DNA in faeces
Front Zool
(2006)
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Estimating bloodstain formation time by quantitative analysis of mtDNA degradation
2022, Forensic Science InternationalCitation Excerpt :Aside from bloodstains, there are relevant studies on the formation time of other types of samples as well. For example, Hara et al. [19] extracted the DNA from old semen samples stored at room temperature, calculated the DNA degradation rate (129:41 and 305:41) based on 41, 129, and 305 bp DNA fragments, and discussed the relationship between the degradation rate and storage time. The results showed that there was no correlation between the DNA degradation rate and storage time.
Effectiveness of whole genome amplification prior to short tandem repeat analysis for degraded DNA
2020, Forensic Science International: GeneticsCitation Excerpt :Specifically, Hara et. al. reported that the number of STR loci detected via the AmpFlSTR Identifiler PCR amplification kit strongly correlated with DI when using 129:41 compared to the 305:41 bp ratio of amplicon quantity, indicating that the former was a more useful index to estimate the number of detectable STR loci [46]. For intact DNA, the DI is near 1, as the quantity of the 129 and 41 bp amplicons is expected to be equivalent.
Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids
2018, Legal MedicineCitation Excerpt :Therefore, the use of ratios reflecting longer fragments (>300 bp) would enable detailed evaluation of the degradation level. In addition to detecting environmental degradation, these ratios were also used to analyze the effect of storage conditions [17]. We used the 10 N DOP-PCR primers with 10 degenerate sequences as a standard.
Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years
2016, Legal MedicineCitation Excerpt :All STR loci were detected in all other bloodstains and blood samples. Hara et al. reported that the Identifiler kit detected ⩽15 loci when the 305:41 bp ratio was ⩽0.0118 [5]. In this study, the 305:41 bp ratio of bloodstains stored at room temperature for 20 years was 0.0220 ± 0.0130 (mean ± standard deviation), and the ratio was 0.015 for the sample with an unamplified D2S1338 locus (data not shown).
Analysis of aged seminal stains by current forensic DNA approach
2015, Forensic Science International: Genetics Supplement SeriesCitation Excerpt :Actually, undetected ‘cold’ cases involving sexual assault can be solved decades after investigations analysing DNA from stored evidences [1]. Recently, STR analysis and the successful typing rate in 19 extremely old seminal stain were assessed [2]. In February 2015, during the restoration work of the museum of the Institute of Legal Medicine at the University of Bologna, three pieces of cotton fabric were found inside an old envelope of the Institute showing handwritten “seminal stains”.