Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples

doi:10.1016/j.legalmed.2015.05.008

Legal Medicine

Volume 17, Issue 5, September 2015, Pages 391-393

https://doi.org/10.1016/j.legalmed.2015.05.008 Get rights and content

Highlights

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DNA degradation ratio correlated moderately with storage duration.
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The detected loci in STR kits correlate with DNA degradation ratio.
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The number of detectable STR loci could be estimated by quantifying two amplicons.

Abstract

The relationships between DNA degradation ratios and the number of detected loci were explored in extremely old seminal stains evaluated using three short tandem repeat (STR) kits: the AmpFlSTR® Identifiler™ PCR Amplification Kit (Identifiler), the AmpFlSTR® Yfiler™ PCR Amplification Kit (Yfiler), and the AmpFlSTR® MiniFiler™ PCR Amplification Kit (MiniFiler). DNA degradation ratios based on 41, 129, and 305 bp DNA fragments were calculated (129:41 and 305:41), and the relationships between the ratios and storage duration were also explored. Using the Identifiler kit, the number of loci detected was strongly correlated with the 129:41 ratio (r = 0.887), whereas the correlation with the 305:41 ratio was moderate (r = 0.656). Using the Yfiler kit, the DYS385 amplicon was detected in all samples, suggesting that DYS385 may be resistant to degradation. The number of detected loci was strongly correlated with the 129:41 ratio (r = 0.768), and moderately so with the 305:41 ratio (r = 0.515). MiniFiler detected at least seven loci in all samples. In samples that did not yield full profiles, the undetected loci were D7S820 and D21S11, or D21S11 only, suggesting that these loci might be easily degraded. The number of loci detected using STR kits correlated with the DNA degradation ratios. In particular, the 129:41 ratio was particularly useful for estimating the number of loci detectable by STR kits. On the other hand, we suggest that storage duration cannot be accurately estimated using DNA degradation ratios; these ratios were not strongly correlated with storage duration (129:41; r = −0.698, 305:41; r = −0.550). However, the ratios may allow the identification of samples that have been stored for more than 40 years.

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.

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Cited by (12)

Estimating bloodstain formation time by quantitative analysis of mtDNA degradation
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Citation 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.
The estimation of bloodstain formation time is still an unsolved problem in forensic science and lacks accurate quantitative methods. Whether DNA can be adopted to estimate bloodstain formation time is still controversial, and there is no study to confirm the potential of mtDNA markers. To address these issues, a triple quantification method based on the ratio of mtDNA fragments of different lengths of COⅠ (mitochondrially encoded cytochrome c oxidase Ⅰ) for estimating bloodstain formation time was established. A total of 152 samples (140 old samples, 12 fresh samples) were collected and tested, and the absolute copies of different-sized fragments of COⅠ (304 bp, 120 bp, 41 bp) in all samples were quantified by SYBR Green real-time qPCR. The natural logarithms of two copy number ratios (304 bp/41 bp, 120 bp/41 bp) of COⅠ in old samples were calculated, which were used as degradation indexes to evaluate the degradation degree of mtDNA. The 140 old human blood samples from 1 to 14 years of storage were accumulated from casework of forensic practice to establish the method of estimating bloodstain formation time and used to analyze the impact of gender factors on the two degradation indexes, and 10 animal samples and 2 fresh human samples were collected to verify the human specificity of the method. There was a high correlation between degradation indexes and bloodstain formation time (the absolute values of correlation coefficients of these two degradation indexes were 0.901 and 0.758 respectively). A method with triple quantification and dual indexes estimating bloodstain formation time was successfully established, which was highly human-specific. There was no statistically significant difference in degradation indexes between different gender samples (P > 0.05). This study confirmed that mtDNA can be utilized to estimate bloodstain formation time, which provides a new solution to the forensic problem of estimating the time of bloodstain formation.
Effectiveness of whole genome amplification prior to short tandem repeat analysis for degraded DNA
2020, Forensic Science International: Genetics
Citation 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.
Short tandem repeat (STR) analysis is prone to failure as DNA is frequently damaged by various environmental factors; hence, increasing the number of starting templates may constitute a feasible approach to improve STR profiling success. Whole genome amplification (WGA) is often applied to bolster starting template quantity. Moreover, WGA can reportedly be used on degraded DNA samples in forensics. Therefore, we utilized a PCR-based WGA method, termed “modified improved primer extension preamplification” (mIPEP), prior to STR analysis of degraded DNA, as this method is less affected by DNA quantity and quality than most others. Saliva from four volunteers was dried on glass fiber filter papers (paper) and glass slides (glass) and irradiated with UVA light (365 nm). The mIPEP method was initiated using 5, 0.5, and 0.05 ng of DNA following DNA extraction. The DNA degradation index (DI) was calculated based on the ratio of 129 to 41 bp DNA fragments; lower numbers indicate higher degradation. Following mIPEP, STR analysis was performed using the AmpFlSTR Identifiler PCR amplification kit. The number of detectable STR loci, with and without mIPEP, decreased according to reduced DI in a different manner for the various DNA concentrations extracted from paper and glass. Specifically, for the 5 ng DNA sample on paper, at a DI < 0.2, the number of detectable STR loci was greater with mIPEP than without it, owing to fewer locus drop-outs. Similarly, the 0.05 ng DNA sample deposited on paper, at DI ≥ 0.7, exhibited higher numbers of detectable STR loci when prepared using mIPEP owing to fewer allele drop-outs. Moreover, among samples deposited on glass, the 0.05 ng DNA sample at DI ≥ 0.4 afforded a larger number of detectable STR loci when prepared using mIPEP than those without mIPEP, owing to fewer locus drop-outs. These findings suggest that performing mIPEP in accordance with sample DNA condition (e.g., quantity and quality) may lead to increased success of STR analysis. Notably, the conditions identified as most responsive to mIPEP were consistent across both UVA-irradiated and environmentally-damaged sample states. Taken together, our results suggest that applying mIPEP would be beneficial to obtain improved STR profiles under conditions involving severely degraded samples with large quantities of DNA, or with small quantities of DNA albeit with slight degradation.
Assessment of DNA degradation of buccal cells under humid conditions and DNA repair by DOP-PCR using locked nucleic acids
2018, Legal Medicine
Citation 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.
We analyzed the degradation level of DNA from buccal cells under humid conditions using quantitative PCR analysis. Gauze samples with buccal cells were incubated for up to 12 months under three different conditions (25 °C/dry, 25 °C/humid, or 40 °C/humid). The degradation was evaluated based on two degradation ratios (129:41 and 305:41 bp). DNA degraded slowly under the 25 °C/humid condition, and significant differences in the two degradation ratios were detected between 25 °C/dry and 25 °C/humid conditions after 12 months. Moreover, the degradation rapidly progressed under the 40 °C/humid condition, and the two degradation ratios in this condition were much lower than those from 25 °C/dry and 25 °C/humid conditions after a short incubation period (3 months). To evaluate the effect of DNA repair on low-copy degraded DNA, degenerate oligonucleotide-primed PCR (DOP-PCR) was performed before short tandem repeats (STR) genotyping. As a standard DOP-PCR, we used a 22-base primer with 10 degenerate sequences (5′-CTCGAGNNNNNNNNNNATGTGG-3′), and additionally designed DOP-PCR primers with 2, 4, 6, or 8 locked nucleic acids (LNAs). When slightly degraded DNA (305:41-bp ratio = 0.60) was used, DOP-PCR significantly increased the fluorescent intensity and success rate of genotyping using Identifiler and Globalfiler kits. In particular, the reaction with four LNAs produced the highest value. However, such benefits were not observed in the analysis of moderately degraded DNA (305:41-bp ratio = 0.13). Although the recovery rates of STR profiles by DOP-PCR were dependent on the degradation level of low-copy DNA, the effectiveness of DOP-PCR highlights the potential of LNA for degenerate sequences.
Effects of storage conditions on forensic examinations of blood samples and bloodstains stored for 20 years
2016, Legal Medicine
Citation 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).
The effects of various storage conditions on blood identification tests, DNA degradation, and short tandem repeat (STR) typing were evaluated. Bloodstains stored at room temperature, 4 °C, −20 °C, and −80 °C for 20 years; blood samples stored at −20 °C and −80 °C for 20 years; and fresh blood samples were analyzed. Leuco-malachite-green testing, anti-human hemoglobin (Hb) testing (using immunochromatography), and tests for hemoglobin-beta (HBB) mRNA were performed as blood identification tests. DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp fragments. STR typing was performed using an AmpFlSTR® Identifiler™ Plus PCR Amplification Kit. All samples were positive in leuco-malachite-green staining and anti-human Hb assays. HBB was not detected in blood samples stored at −20 °C or −80 °C, although this marker was detected in all bloodstains. As indicated by the ratio of 129:41 bp and 305:41 bp DNA fragments, DNA from bloodstains stored at room temperature or 4 °C were significantly degraded compared to DNA from all other samples. STR typing analyses revealed that a portion of the loci was undetected in bloodstains stored at room temperature. Therefore, to prevent DNA degradation during long-term storage, it is recommended that bloodstains and blood be stored at below −20 °C. In addition, because bloodstains are more suitable for detection of blood-specific mRNAs than blood sample, it is desirable that blood is stored as bloodstain for this method.
Analysis of aged seminal stains by current forensic DNA approach
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Citation 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”.
‘Cold’ cases involving sexual assault can be solved decades after investigations analysing DNA from stored evidences, collected when current forensic method were not developed. To evaluate the influence of time and storage conditions on presumptive test and DNA analysis, three aged seminal stains date back to the first half of the ‘900, recovered during restoration work of the Institute of Legal Medicine of the University of Bologna, were analysed by the semenogelin test, microscopic identification of spermatozoa, autosomal, Y-chromosome and X chromosome STRs, and mitochondrial DNA analysis. All samples displayed a weak positive reaction for semenogelin test and the sperm heads were clearly identified. The number of detectable autosomal and Y-STRs was strongly correlated to the DNA degradation index, except for one specimen showing a high degradation index but enough DNA to obtain a composite profile. STRs included in the Minifiler kit and smaller STRs in ESX System were reproducible in all analysed specimens. Aged seminal stains may be successfully analysed by current forensic methods, even if the selection of appropriate amplicons size based on DNA amount and its degradation index is mandatory to predict the success probability in casework.
Using Human DNA from Environmental Water to Identify People and the Location of a Drowning Site
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View full text

Short CommunicationRelationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples

Highlights

Abstract

Introduction

Section snippets

Samples

DNA extraction and quantification

Relationships between DNA degradation ratios and storage duration

Conclusion

Acknowledgments

Evaluation of forensic examination of extremely aged seminal stains

Legal Med

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies

Forensic Sci Int

A quantitative PCR assay for the assessment of DNA degradation in forensic samples

Forensic Sci Int

Quantification of damage in DNA recovered from highly degraded samples–a case study on DNA in faeces

Front Zool

Short Communication
Relationship between DNA degradation ratios and the number of loci detectable by STR kits in extremely old seminal stain samples