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Kim S.W.,Korea University | Heo J.H.,Korea University | Kim C.H.,Korea University | Yoo D.C.,Korea University | And 7 more authors.
Journal of Toxicology and Environmental Health - Part A: Current Issues | Year: 2010

Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods. Copyright © Taylor & Francis Group, LLC. Source


Cha Y.S.,Research and Development Center | Choi S.H.,Research and Development Center | Lee J.-H.,Research and Development Center | Shin S.-K.,Research and Development Center | And 4 more authors.
Analytical Biochemistry | Year: 2011

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI-TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI-TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders. © 2011 Elsevier Inc. Source


Kim T.W.,Korea Research Institute of Bioscience and Biotechnology | Kim T.W.,Chungnam National University | Kang Y.K.,Inje University | Park Z.Y.,Gwangju Institute of Science and Technology | And 14 more authors.
Carcinogenesis | Year: 2014

SH3RF (SH3-domain-containing RING finger protein) family members, SH3RF1-3, are multidomain scaffold proteins involved in promoting cell survival and apoptosis. In this report, we show that SH3RF2 is an oncogene product that is overexpressed in human cancers and regulates p21-activated kinase 4 (PAK4) protein stability. Immunohistochemical analysis of 159 colon cancer tissues showed that SH3RF2 expression levels are frequently elevated in cancer tissues and significantly correlate with poor prognostic indicators, including increased invasion, early recurrence and poor survival rates. We also demonstrated that PAK4 protein is degraded by the ubiquitin-proteasome system and that SH3RF2 inhibits PAK4 ubiquitination via physical interactionmediated steric hindrance, which results in the upregulation of PAK4 protein. Moreover, ablation of SH3RF2 expression attenuates TRADD (TNFR-associated death domain) recruitment to tumor necrosis factor-α (TNF-α) receptor 1 and hinders downstream signals, thereby inhibiting NF-κB (nuclear factor-kappaB) activity and enhancing caspase-8 activity, in the context of TNF-α treatment. Notably, ectopic expression of SH3RF2 effectively prevents apoptosis in cancer cells and enhances cell migration, colony formation and tumor growth in vivo. Taken together, our results suggest that SH3RF2 is an oncogene that may be a definitive regulator of PAK4. Therefore, SH3RF2 may represent an effective therapeutic target for cancer treatment. © The Author 2013. Published by Oxford University Press. All rights reserved. Source


Tomas C.,Copenhagen University | Skitsa I.,DNA Analysis Laboratory | Steinmeier E.,Copenhagen University | Poulsen L.,Copenhagen University | And 3 more authors.
Forensic Science International: Genetics | Year: 2015

A population sample of 223 Greek individuals was typed for five sets of forensic genetic markers with the kits NGM SElect™, SNPforID 49plex, DIPplex®, Argus X-12 and PowerPlex® Y23. No significant deviation from Hardy-Weinberg expectations was observed for any of the studied markers after Holm-Šidák correction. Statistically significant (P < 0.05) levels of linkage disequilibrium were observed between markers within two of the studied X-chromosome linkage groups. AMOVA analyses of the five sets of markers did not show population structure when the individuals were grouped according to their geographic origin. The Greek population grouped closely to the other European populations measured by FST∗ distances. The match probability ranged from a value of 1 in 2 × 107 males by using haplotype frequencies of four X-chromosome haplogroups in males to 1 in 1.73 × 1021 individuals for 16 autosomal STRs. © 2015 Elsevier Ireland Ltd. All rights reserved. Source


Seo S.B.,Seoul National University | Lee H.Y.,Seoul National University | Zhang A.H.,Seoul National University | Kim H.Y.,DNA Analysis Laboratory | And 3 more authors.
International Journal of Legal Medicine | Year: 2012

Correct DNA quantification is an essential part to obtain reliable STR typing results. Forensic DNA analysts often use commercial kits for DNA quantification; among them, real-time-based DNA quantification kits are most frequently used. Incorrect DNA quantification due to the presence of PCR inhibitors may affect experiment results. In this study, we examined the alteration degree of DNA quantification results estimated in DNA samples containing a PCR inhibitor by using a Quantifiler® Human DNA Quantification kit. For experiments, we prepared approximately 0.25 ng/μl DNA samples containing various concentrations of humic acid (HA). The quantification results were 0.194-0.303 ng/μl at 0-1.6 ng/μl HA (final concentration in the Quantifiler reaction) and 0.003- 0.168 ng/μl at 2.4-4.0 ng/μl HA. Most DNA quantity was undetermined when HA concentration was higher than 4.8 ng/μl HA. The CT values of an internal PCR control (IPC) were 28.0-31.0, 36.5-37.1, and undetermined at 0- 1.6, 2.4, and 3.2 ng/μl HA. These results indicate that underestimated DNA quantification results may be obtained in the DNA sample with high CT values of IPC. Thus, researchers should carefully interpret the DNA quantification results. We additionally examined the effects of HA on the STR amplification by using an Identifiler® kit and a MiniFiler kit. Based on the results of this study, it is thought that a better understanding of various effects of HA would help researchers recognize and manipulate samples containing HA. © Springer-Verlag 2011. Source

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