DNA-Based Identification of Necrophagous Fly Species Using Abdominal-B (Abd-B) Homeobox Sequence / 대한법의학회지

In medicolegal investigations, correct identification of the necrophagous fly species collected around and on the corpse is an essential step for estimating the postmortem interval (PMI). Therefore, forensic pathologists and entomologists investigating deaths due to violent crimes need a rapid, easy-to-use protocol to identify fly species found on corpses. A rapid and robust DNA-based tool that can distinguish between various immature and mature species from the Calliphoridae, Muscidae, and Sarcophagidae families would be ideal for such investigations. To date, the DNA barcode initiative is the best approach for identifying species-specific nucleotide sequences. We have developed 3 sequence-characterized amplified region (SCAR)-based identification systems derived from the Abdominal-B homeobox sequences of 17 fly species belonging to the Muscidae and Sarcophagidae. The flies used in this study were collected in Korea. These assay systems can classify 17 forensically important fly species into the dipteran family group and reliably distinguish them from inter- and intraspecific fly species through a 2-step multiplex PCR. This novel approach may also be used as an alternative to conventional DNA-based identification methods.

Use of Cytochrome c Oxidase Subunit I (COI) Nucleotide Sequences for Identification of the Korean Luciliinae Fly Species (Diptera: Calliphoridae) in Forensic Investigations

Blowflies, especially species belonging to the subfamily Luciliinae, are the first insects to lay eggs on corpses in Korea. Fast and accurate species identification has been a key task for forensic entomologists. Because conventional morphologic identification methods have many limitations with respect to forensic practice, molecular methods have been proposed to identify fly species of forensic importance. To this end, the authors amplified and sequenced the full length of the cytochrome c oxidase subunit I (COI) gene of the Luciliinae fly species collected in Korea. The results showed the COI sequences are instrumental in identifying Luciliinae fly species. However, when compared with previously reported data, considerable inconsistencies were noted. Hemipyrellia ligurriens data in this study differed significantly from two of the five pre-existing data. Two closely related species, Lucilia illustris and Lucilia caesar, showed an overlap of COI haplotypes due to four European sequences. The results suggest that more individuals from various geographic regions and additive nuclear DNA markers should be analyzed, and morphologic identification keys must be reconfirmed to overcome these inconsistencies.

Evaluation of Nine Non-CODIS MiniSTR Loci to aid Analysis of Degraded DNA / 대한법의학회지

For highly degraded DNA samples of forensic casework, new miniSTR PCR systems have been developed to supplement the current CODIS STRs. In the present study, we established the three miniplexes for nine miniSTRs (NC01 : D10S1248, D14S1434 and D22S1045; NC02 : D1S1677, D2S441 and D4S2364; and NC03 : D3S3053, D6S474 and D20S482) which had been previously suggested by Butler group (NIST, Gaitherburg, MD, USA). To evaluate the usefulness of the nine miniSTRs in analysis of degraded DNA, the sensitivity and efficacy of the three miniplexes were determined and then compared with those of the BigMini STR system which consists of six CODIS miniSTRs (TH01, CSF1PO, FGA, TPOX, D7S820, and D21S11). The three miniplexes gave better results in both the sensitivity test and efficiency test in comparison with BigMini. In the sensitivity test using serially diluted standard DNA, most loci in the three miniplexes showed reliable results for samples containing 50 pg of DNA and some even showed good sensitivity for samples containing 30 pg of DNA. Additionally, the three miniplexes generated useful profiles for both enzymatically degraded DNA and 50-year old skeletal remain samples. Among the nine miniSTRs, D4S2364, D3S3053, D14S1434, and D1S1677 produced the most successful DNA profiles for old skeletal remains. These results suggest that new miniSTRs could be useful supplements to the 13 CODIS STRs for forensic analysis of degraded DNA.

Haplotypes and mutations of 17 Y-STR loci from Korean father-son pairs / 대한법의학회지

We have investigated 17 Y-STR loci (DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 (Y GATA C4), Y GATA H4) in 365 Korean father-son pairs of 355 families. Of 338 different haplotypes obtained from 355 fathers, 326 haplotypes were observed once, 10 haplotypes two times and the other two haplotypes were observed 4 and 5 times, respectively. The overall haplotype diversity was 0.9996. In 365 father-son pairs, a total of 21 mutations were observed at 12 Y-STR loci. Sequence analysis for mutant alleles demonstrated 21 single step mutations: 8 gains and 13 losses. However, there was no significant surplus of gains or losses. The locus-specific mutation rate estimates were between 0.0 and 8.2 x 10(-3) and the average mutation rate estimates were 3.4 x 10(-3)(95% C.I. 2.1-5.2 x 10(-3)) across all 17 Y-STR loci.

A Population Study of X-chromosomal STRs, DXS9898, DXS6809, DXS7424 and DXS10011 in Koreans / 대한법의학회지

A population study of the X-chromosomal short tandem repeat (STR) loci DXS9898, DXS6809, DXS7424 and DXS10011 was carried out by single multiplex PCR in a sample of 300 unrelated Korean individuals (150 males and 150 females). For accurate and reproducible STR typing, sequenced allelic ladders were constructed and GenoTyper macro was programmed. In this study, four types of the repre-sentative repeat sequence structure of DXS10011 were observed and the allele loss at DXS9898 was observed in 13 of 450 chromosomes (2.9%). The inter-population comparison of the allele frequencies at the 4 X-STRs showed significantly different distributions (p<0.01) for Koreans and Germans except DXS10011. All statistical parameters for forensic efficiency showed that the 4 X-chromosomal STRs are highly informative. Especially, DXS10011 is expected to be the most useful marker for forensic practice.

Simple Quantification and DNA Profiling from Degraded Low Copy Number DNA Samples / 대한법의학회지

DNA quantification is important to ensure the consistency and the reliability in the interpretation of degraded low copy number DNA typing. We applied the simple PCR quantification method using fluo-rescently labeled primers for the amplification of mtDNA and amelogenin gene in 50 year old skeletal remains (e.g. bone and tooth). K562 DNA was serially diluted and used as a standard for concentration marker to gauge the amount of DNA from PCR versus the peak area. The quantities of DNA extracted from bones and teeth did not show significant difference in the analyses both using mtDNA and amelo-genin gene as an amplification target. To test the efficiency of DNA profiling of degraded low copy number DNA samples, mtDNA PCR quality evaluation and DNA typing for 16 autosomal STR and 9 Y chromosomal STR loci were per-formed and the correlation between DNA quantities and PCR amplification efficiencies of the samples was analyzed. The DNA quantities assayed by the simple method suggested in the present study could be good indicator for mtDNA and STR analysis. As the allele drop-out was observed in less than 0.050ng DNA samples, at least 0.100ng of DNA is required to produce informative STR profiles. Also, STRs with less than 200bp amplification sizes produce efficient DNA profiles in most cases. Therefore, the develop-ment of mini-STRs with less than 200bp amplification sizes is expected to improve DNA typing in degraded low copy number DNA. Y-STRs are easy to detect allele drop-out or drop-in, and accordingly the efficiency test of Y-STRs as well as autosomal STRs for profiling of degraded low copy number DNA samples is thought to be important.

Intra-individual Difference of Length Heteroplasmy in Blood and Hair Shaft Mitochondrial DNA / 대한법의학회지

To observe mtDNA length heteroplasmy in a homoploymeric cytosine tract of the mitochondrial HV2 region, we carried out size-based separation of PCR products, which was produced by using primers designed to minimize the stutter production. Blood and hair shaft samples were collected from 25 individuals. The result showed significant qualitative/quantitative peak pattern variations among blood and hair shaft mtDNA profiles. Based on the results of this study, an exclusion depended solely on differences in length of the major C-tract variant could thus be an erroneous interpretation. Therefore, differences in the number of cytosine or qualitative/quantitative peak pattern variations in the C-tract of the mtDNA HV2 region cannot be used alone to support an interpretation of exclusion.