Application of hematoxylin reagent for sperm cell separation in sexual crime evidence.

Seminal evidence obtained from a sexual crime scene provides clues for solving a case through forensic analysis. However, most evidence collected from sexual crime scenes is a mixture of sperm cells and vaginal discharge. Therefore, separating the sperm cells from the seminal evidence is very important. In this study, we developed a separation method for effectively separating sperm cells using differential extraction with commercially available sperm staining reagents such as hematoxylin and nigrosin. Hematoxylin (0.03 % v/v) effectively stained the sperm cells in ATL and TNE lysis buffer, while nigrosin did not. The loss of sperm cells during washing of the specimen was minimized using the differential extraction method. Subsequently, genomic DNA was extracted from the hematoxylin-stained sperm cells and subjected to short tandem repeat genotyping. We observed no interference from hematoxylin. These results indicate that hematoxylin can be used to stain sperm cells and thus facilitate subsequent genetic identification.

Simplified Direct PCR Method for Reference Buccal Samples Using a Non-FTA Card by Omitting the Pretreatment Step.

When using non-FTA cards in commercial multiplex STR kits for direct PCR, pretreatment steps with specific buffers are recommended. Here, we designed a rapid direct PCR method utilizing a non-FTA card, Oral Cell Sampling Kit, by omitting the pretreatment step involving Prep-n-Go™ Buffer, and it showed compatibility with the GlobalFiler™ Express PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit, and PowerPlex® Fusion system. To optimize the PCR conditions, we tested the method with different final PCR volumes and cycles. Finally, we conducted a performance test using 50 Korean buccal samples and confirmed the high performance of the method, detecting more than 90% of the samples with full profiles when using GlobalFiler™ PCR Amplification Kit and PowerPlex® Fusion system at 29 cycles in a 10 µL final PCR volume. Thus, we report a simple direct PCR set-up to analyze reference samples collected using a non-FTA card manufactured in Korea.

Ancestry informative markers (AIMs) for Korean and other East Asian and South East Asian populations.

Inference of ancestry from biological evidence can provide investigative information, especially for unknown DNA donors. Although tools for predicting ancestry have been developing, ancestry research focusing on populations relevant for South Korea is not common and markers are seldom chosen specifically to differentiate Koreans from other East Asian and South East Asian populations. Here, we report ancestry informative markers (AIMs) for distinguishing six East/South East Asian regional populations: China, Japan, Indonesia, Philippines, South Korea and Thailand. Individual genotypes from these six populations were available in PanSNPdb: The HUGO Pan-Asian SNP Database. To select AIMs, we calculated four population divergence metrics for each SNP: Nei's FST, Rosenberg's Informativeness (In), the average absolute allele frequency difference between populations (δFmean) and the maximum allele frequency difference between populations (δFmax). Based on these values, we selected 100 single nucleotide polymorphisms (SNPs) for distinguishing the six populations, 13 of which exhibited large allele frequency differences between Koreans and non-Koreans. To assess the performance of the AIMs, we performed principal coordinates analysis (PCoA) on the individuals from all six populations and inferred ancestral population clusters using the STRUCTURE program. In conclusion, we found that the selected AIMs can be applied to distinguish the six East/South East Asian groups and we suggest the markers in this study will be helpful to establish ancestry panels for Korea and neighbouring populations.

Identification of female-specific blood stains using a 17ß-estradiol-targeted aptamer-based sensor.

Blood stain evidence obtained from a violent crime scene provides decisive clues that can enable a case to be solved through forensic analyses such as genetic identification. However, collected samples usually contain a mixture of biological material from different sources, making genetic identification difficult. To address this issue, we developed an activatable aptamer sensor targeting 17ß-estradiol for detection of female-specific blood in mixed samples. With the sensor, we were able to detect blood originating from females using a variable light source (495 nm). The sensor was especially sensitive to blood from young females (10-40 years) but not to blood from older females (≥ 50 years). Genomic DNA was extracted from the female blood specimens identified by this method and used for quantification and short tandem repeat genotyping. We confirmed that there was no fluorescence interference from the aptamer sensor. These results indicate that this novel aptamer sensor can be used to analyze evidentiary blood samples and thereby facilitate subsequent genetic identification.

Development of real-time PCR assay for genetic identification of the mottled skate, Beringraja pulchra.

The mottled skate, Beringraja pulchra is one of the commercially important fishes in the market today. However, B. pulchra identification methods have not been well developed. The current study reports a novel real-time PCR method based on TaqMan technology developed for the genetic identification of B. pulchra. The mitochondrial cytochrome oxidase subunit 1 (COI) nucleotide sequences of 29 B. pulchra, 157 skates and rays reported in GenBank DNA database were comparatively analyzed and the COI sequences specific to B. pulchra was identified. Based on this information, a system of specific primers and Minor Groove Binding (MGB) TaqMan probe were designed. The assay successfully discriminated in 29 specimens of B. pulchra and 27 commercial samples with unknown species identity. For B. pulchra DNA, an average Threshold Cycle (Ct) value of 19.1±0.1 was obtained. Among 27 commercial samples, two samples showed average Ct values 19.1±0.0 and 26.7±0.1, respectively and were confirmed to be B. pulchra based on sequencing. The other samples tested showed undetectable or extremely weak signals for the target fragment, which was also consistent with the sequencing results. These results reveal that the method developed is a rapid and efficient tool to identify B. pulchra and might prevent fraud or mislabeling during the distribution of B. pulchra products.

Population genetics of insertion-deletion polymorphisms in South Koreans using Investigator DIPplex kit.

We assessed the applicability of 30 insertion-deletion polymorphisms (INDELs) in forensic use and the level of genetic diversity in South Korea (n=373) using the Investigator DIPplex kit (Qiagen). Allele frequencies, heterozygocities, and forensic efficacy parameters were determined. No deviation from Hardy-Weinberg equilibrium was observed for any of the INDEL markers. A high level of discrimination power was observed (combined power of discrimination: 0.99999999995). The combined match probability value was 2.84 × 10(-11) and the mean typical paternity indices were 0.878. Furthermore, we found one microvariant allele at HLD93 (rs2307570) that has not been reported. We expect that these 30 loci of INDEL markers will be useful for forensic identification and paternity testing in the South Korean population.