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.

Identification of Korean-specific SNP markers from whole-exome sequencing data.

Analysis of large numbers of single-nucleotide polymorphisms (SNPs) can increase individual discrimination power, and, particularly, it can supply important evidence for kinship or ethnic identification. We identified 300 Korean-specific SNPs from 306 Korean whole-exome sequencing (WES) data. Functionally significant SNPs (variants in splicing site, missense, nonsense, and exonic indels) were filtered out from the variant pool, and SNPs with minor allele frequencies (MAFs) of <0.3 in the 1000 Genomes (1000G) database but >0.3 in the Korean population were selected. Genotypes obtained from WES were confirmed by the Sanger sequencing method. The identified markers were evenly distributed throughout the autosomal chromosomes. All the SNPs were in the Hardy-Weinberg equilibrium with a mean MAF of 0.415 (0.161 in 1000G). The mean heterozygosities were 0.476 (observed) and 0.470 (experimental). The combined power of discrimination was very high. Korean MAFs in most SNPs were similar to those for the Chinese and Japanese populations, but were significantly higher than those for several other ethnic populations. These selected SNPs will be used to develop forensic markers and are expected to be widely used for additional individual identification, ethnic discrimination, and linkage analysis for kinship tests.

Evaluation of samples comprising minute amounts of DNA.

Minute amounts of DNA representing only few diploid cells, may be interrogated using enhanced DNA profiling, which will be accompanied by stochastic amplification effects. Notwithstanding, a weight of evidence statistic may be calculated using current interpretation software. In this study, we profiled single donor, two- and three-person samples having only 3 pg to 12 pg of DNA per contributor using both standard and enhanced capillary electrophoresis (CE) injection settings. Likelihood ratios (LRs) were computed using LRmix Studio, compared for both types of profiles and examined in relation to the amount of DNA, drop-out level, number of detected alleles, peak heights and reproducibility of alleles. Especially for DNA profiles that were generated using enhanced CE, the obtained LRs could indicate strong evidence in favour of the prosecution (log10(LR)>6), also when the amount of DNA represented about half of a diploid cell equivalent in the amplification. These results illustrate that an assessment of the criminalistic relevance of a sample carrying minute amounts of DNA is essential prior to applying enhanced interrogation techniques and/or calculating a weight of evidence statistic.

A large population genetic study of 15 autosomal short tandem repeat loci for establishment of Korean DNA profile database.

Genotyping of highly polymorphic short tandem repeat (STR) markers is widely used for the genetic identification of individuals in forensic DNA analyses and in paternity disputes. The National DNA Profile Databank recently established by the DNA Identification Act in Korea contains the computerized STR DNA profiles of individuals convicted of crimes. For the establishment of a large autosomal STR loci population database, 1805 samples were obtained at random from Korean individuals and 15 autosomal STR markers were analyzed using the AmpFlSTR Identifiler PCR Amplification kit. For the 15 autosomal STR markers, no deviations from the Hardy-Weinberg equilibrium were observed. The most informative locus in our data set was the D2S1338 with a discrimination power of 0.9699. The combined matching probability was 1.521 × 10(-17). This large STR profile dataset including atypical alleles will be important for the establishment of the Korean DNA database and for forensic applications.

Population genetic polymorphisms of 17 Y-chromosomal STR loci in South Koreans.

Haplotypes and allele frequencies of 17 Y-chromosomal STR loci included in the AmpFlSTR(®) Yfiler(®) system were obtained from a sample of 1021 unrelated individuals living in 6 provinces of South Korea. A total of 938 haplotypes were observed in the 1021 individuals studied, of which 885 were unique. The overall haplotype diversity for the 17 Y-STR loci was 0.9998, and the discrimination capacity was 0.9187. We found 11 atypical alleles (null, duplicated, triplicated, and microvariant alleles), that have not previously been reported in South Korean populations. It seems that these 17 loci are useful genetic markers for forensic personal identification and paternity testing in the South Korean population.

Fine particle removal performance of a two-stage wet electrostatic precipitator using a nonmetallic pre-charger.

A novel two-stage wet electrostatic precipitator (ESP) has been developed using a carbon brush pre-charger and collection plates with a thin water film. The electrical and particle collection performance was evaluated for submicrometer particles smaller than 0.01- 0.5 micrometer in diameter by varying the voltages applied to the pre-charger and collection plates as well as the polarity of the voltage. The collection efficiency was compared with that calculated by the theoretical models. The long-term performances of the ESP with and without water films were also compared in tests using Japanese Industrial Standards dust. The experimental results show that the carbon brush pre-charger of the two-stage wet ESP had approximately 10% particle capture, while producing ozone concentrations of less than 30 ppb. The produced amounts of ozone are significantly lower than the current limits set by international agencies. The ESP also achieved a high collection rate performance, averaging 90% for ultrafine particles, as based on the particle number concentration at an average velocity of 1 m/sec corresponding to a residence time of 0.17 sec. Higher particle collection efficiency for the ESP can be achieved by increasing the voltages applied to the pre-charger and the collection plates. The decreased collection efficiency that occurred during dust loading without water films was completely avoided by forming a thin water film on the collection plates at a water flow rate of 6.5 L/min/m(2).

Production and characterization of active hepatitis C virus RNA-dependent RNA polymerase.

The non-structural protein 5B (NS5B) is an essential component for the genome replication of hepatitis C virus (HCV). Thus, its activity holds the potential of being a target for therapeutic actions against HCV. The availability of large amount of functionally active NS5B enzyme may facilitate the identification of NS5B inhibitors via high-throughput screening (HTS). Here, we expressed the C-terminal 20-amino acids truncated NS5B in a bacterial system using the N-terminal domain of Escherichia coli lysyl-tRNA synthetase (LysN) as a solubility enhancer. The fusion protein (LysN-NS5B) was purified in a yield of 6.2mg/L. The activity of LysN-NS5B was confirmed by in vitro RNA-dependent RNA polymerase (RdRp) activity assay, and the biochemical properties of LysN-NS5B were further characterized by kinetic analysis. The optimal RdRp activity was shown at 30 degrees C with 5mM of Mg(2+) or 10mM of Mn(2+), while the K(m) value for UTP was determined as 5microM. The RdRp activity of LysN-NS5B was strongly inhibited by phenyldiketoacid, a specific inhibitor of HCV NS5B activity. Our results suggest that the LysN fusion system is a suitable approach for producing an active form of NS5B that can be used for HTS of NS5B inhibitors.