• Journal of Oral Medicine and Pain
• /
• v.24 no.3
• /
• pp.335-345
• /
• 1999

법의학적 개인식별 및 친생자 감정시 여러 개의 single tandem repeats(STR) 유전좌위 검색이 필요하다. 그 이유는 STR 유전좌위는 대립유전자 수가 적고 이형접합도가 낮아 서로 다른 개체간에도 동일한 유전좌위를 가질 확률이 높기 때문에 개인식별에 대한 기여도가 떨어지게 된다. 따라서 여러 개의 다양한 STR 유전좌위들을 동시에 분석함으로써 우연적으로 개체간에 유전자형이 일치할 가능성을 낮추어야 감정의 신뢰성을 높일 수 있으며 이에는 각 STR 유전좌위에 대한 유전좌위의 분포가 인종별, 지역별로 달라 이에 대한 유전자분포를 구하는 것이 선행조건이다. 이에 본 연구에서는 법의학적 개인식별 및 친자감정시 기초자료로 활용하기 위하여 서로 혈연관계가 없는 201명의 한국인 혈액에서 DNA를 추출하여 STR 유전좌위증 human coagulation factor XIII A subunit gene(F13A0l Locus), human c-fes/fps proto-oncogene(FESFPS Locus), human von Willebrand factor gene (vWA Locus)등 FFv Triplex 유전자를 중합효소반응에 의하여 동시에 증폭하고, 폴리아크릴아마이드겔을 이용한 전기영동 및 질산은 염색을 시행한 후 FFv Triplex유전자의 유전자형 및 대립유전자 빈도 등을 분석하여 다음과 같은 결과를 얻었다. (1) F13A01유전자는 5개의 대립유전자, 12개의 유전자형을 검출하였으며, 이형접합도는 60.7%로 나타났고 대립유전자 및 유전자빈도는 3.2, 4, 5, 6, 16 대립유전자에서 각각 0.34 3, 0.114, 0.062, 0.475, 0.005로 나타났으며, 대립유전자 7, 8, 9, 10, 11, 12, 13, 14, 15는 검출되지 않았다. (2) F13A01 대립유전자다양성 (allelic diversity value)은 0.641, 개인식별력(PD)은 0.814를 보였으며 대립유전자다양성 및 이형접합도가 다른 민족과 비교할 때 다소 낮았다. (3) FESFPS유전자는 8개 대립유전자 모두 나타났으며, 15개의 유전자형을 검출하였으며, 이형접합도는 66.7%로 나타났고 대립유전자 및 유전자빈도는 7, 8, 9, 10, 11, 11, 12, 13, 14 대립유전자에서 각각 0.002, 0.002, 0.005, 0.032, 0.507, 0.264, 0.197, 0.007로 나나났다. (4) FESFPS 대립유전자다양성(allelic diversity value)은 0.641, 개인식별력(PD)은 0.804를 보였다. (5) vWA유전자는 9개의 대립유전자, 23개의 유전자형을 검출하였으며, 이형접합도는 80.1%로 나타났고 대립유전자 및 유전자 빈도는 11, 12, 14, 15, 16, 17, 18, 19, 20 대립유전자 에서 각각 0.002, 0.002, 0.219, 0.032, 0.187, 0.279, 0.189, 0.072, 0.017로 나타났으며, 대립 유전자 13, 21는 검출되지 않았다. (6) vWA 대립유전자다양성(allelic diversity value)은 0.799, 개인식별력(PD)은 0.924로 매우 높게 나타냈다.

• Journal of the Korea Institute of Military Science and Technology
• /
• v.27 no.4
• /
• pp.507-515
• /
• 2024

Microbial forensics is a scientific discipline for analyzing evidence related to biological crimes by identifying the origin of microorganisms. Multiple locus variable number tandem repeat analysis(MLVA) is one of the microbiological analysis methods used to specify subtypes within a species based on the number of tandem repeat in the genome, and advances in next generation sequencing(NGS) technology have enabled in silico anlysis of full-length whole genome sequences. In this paper, we analyzed unknown samples provided by Robert Koch Institute(RKI) through The United Nations Secretary-General's Mechanism(UNSGM)'s external quality assessment exercise(EQAE) project, which we officially participated in 2023. We confirmed that the 3 unknown samples were B. anthracis through nucleic acid isolation and genetic sequence analysis studies. MLVA results on 32 loci of B. anthracis were analysed by using genome sequences obtained from NGS(NextSeq and MinION) and Sanger sequencing. The MLVA typing using short-reads based NGS platform(NextSeq) showed a high probability of causing assembly error when a size of the tandem repeats was grater than 200 bp, while long-reads based NGS platform(MinION) showed higher accuracy than NextSeq, although insertion and deletion was observed. We also showed hybrid assembly can correct most indel error caused by MinION. Based on the MLVA results, genetic identification was performed compared to the 2,975 published MLVA databases of B. anthracis, and MLVA results of 10 strains were identical with 3 unkonwn samples. As a result of whole genome alignment of the 10 strains and 3 unknown samples, all samples were identified as B. anthracis strain A4564 which is associated with injectional anthrax isolates in heroin users.

• Journal of Genetic Medicine
• /
• v.7 no.1
• /
• pp.59-66
• /
• 2010

Purpose: Quantitative fluorescent polymerase chain reaction (QF-PCR) allows for the rapid prenatal diagnosis of common aneuploidies. The main advantages of this assay are its low cost, speed, and automation, allowing for large-scale application. However, despite these advantages, it is not a routine method for prenatal aneuploidy screening in Korea. Our objective in the present study was to validate the performance of QF-PCR using short tandem repeat (STR) markers in a Korean population as a means for rapid prenatal diagnosis. Material and Methods: A QF-PCR assay using an Elucigene kit (Gen-Probe, Abingdon, UK), containing 20 STR markers located on chromosomes 13, 18, 21, X and Y, was performed on 847 amniotic fluid (AF) samples for prenatal aneuploidy screening referred for prenatal aneuploidy screening from 2007 to 2009. The results were then compared to those obtained using conventional cytogenetic analysis. To evaluate the informativity of STR markers, the heterozygosity index of each marker was determined in all the samples. Results: Three autosomes (13, 18, and 21) and X and Y chromosome aneuploidies were detected in 19 cases (2.2%, 19/847) after QF-PCR analysis of the 847 AF samples. Their results are identical to those of conventional cytogenetic analysis, with 100% positive predictive value. However, after cytogenetic analysis, 7 cases (0.8%, 7/847) were found to have 5 balanced and 2 unbalanced chromosomal abnormalities that were not detected by QF-PCR. The STR markers had a slightly low heterozygosity index (average: 0.76) compared to those reported in Caucasians (average: 0.80). Submicroscopic duplication of D13S634 marker, which might be a unique finding in Koreans, was detected in 1.4% (12/847) of the samples in the present study. Conclusion: A QF-PCR assay for prenatal aneuploidy screening was validated in our institution and proved to be efficient and reliable. However, we suggest that each laboratory must perform an independent validation test for each STR marker in order to develop interpretation guidelines of the results and must integrate QF-PCR into the routine cytogenetic laboratory workflow.

• Korean Journal of Microbiology
• /
• v.43 no.4
• /
• pp.243-249
• /
• 2007

The centromere is a highly differentiated structure of the chromosome that fulfills a multitude of essential mitotic and meiotic functions. Alphoid DNA (${\alpha}$-satellite) is the most abundant family of repeated DNA found at the centromere of all human chromosomes, and chromosomes of primates in general. The most important parts in the development of Human Artificial Chromosomes (HACs), are the isolation and maintenance of stability of centromeric region. For isolation of this region, we could use the targeting hook with alphoid DNA repeat and cloned by Transformation-Associated Recombination (TAR) cloning technique in yeast Saccharomyces cerevisiae. The method includes rolling-circle amplification (RCA) of repeats in vitro to 5 kb-length and elongation of the RCA products by homologous recombination in yeast. Four types of $35\;kb{\sim}50\;kb$ of centromeric DNA repeat arrays (2, 4, 5, 6 mer) are used to examine the stability of repeats in homologous recombination mutant strains (rad51, rad52, and rad54). Following the transformation into wild type, rad51 and rad54 mutant strains, there were frequent changes in inserted size. A rad52 mutant strain showed extremely low transformation frequency, but increased stability of centromeric DNA repeat arrays at least 3 times higher than other strains. Based on these results, the incidence of large mutations could be reduced using a rad52 mutant strain in maintenance of centromeric DNA repeat arrays. This genetic method may use more general application in the maintenance of tandem repeats in construction of HAC.

• Proceedings of the Korea Society of Poultry Science Conference
• /
• 2005.11a
• /
• pp.64-65
• /
• 2005

Members of the Pumilio are the RNA binding proteins acting as translational repressors and required for germ cell development and asymmetric division. We identified chicken Pum1 and Pum2 that are similar to mouse and human in highly conserved C-terminal RNA-binding domain and eight tandem repeats. The comparative sequence analysis of Pum1 and Pum2 from fly, chicken, mouse and human shows high degree of evolutionary conservation in the homology of the peptide sequence and the structure of PUM-HD (Pumilio homology domain) with similar spacing between adjacent Pum repeats. Also, structures of chicken Pum1 and Pum2 genes are almost identical to those of mouse and human. We revealed that the expression levels of Pum1 and Pum2 were the highest in hatched female gonad among various embryonic tissues, and Pum2 expressed highly in 12-day and hatched gonad by real-time RT-PCR. These results suggest that Pum1 and Pum2 might have an effect on the development of chicken gonad.

• The Korean Journal of Zoology
• /
• v.28 no.3
• /
• pp.166-178
• /
• 1985

In order to express hepatitis B surface antigen $(HB_sAg)$ containing pre-surface antigen region in mammalian calls, 2.7 kb DNA fragment containing pre-surface region-$HB_sAg$ gene poly(A) addition site of HBV genome was cloned into simian virus 40(SV 40) based chimeric vector pSVOB. 2.7 kb DNA fragment was derived from pHBVD 107 containing tandem copies of the HBV genome in a head-to-tail arrangement by Bgl II digestion. Construction of the vector pSVOE involved the incorporation of SV40 sequences spanning the viral origin of replication and 72 bp repeats (enhancer) into a pBR 322 derivative lacking sequences which inhibit replication in mammalian cells. Bam HI linker was inserted at the Pvu II site in the proximity of SV40 late promoter of pSVOE and named as pSVOB. To construct the recombinant plasmid pSVBS, pHBVD 107 was digested with Bgl II to isolate 2.7kb DNA fragment and the fragment was ligated into the Bam HI site of pSVOB by ligation. Preliminary result showed that the recombinant plasmid pSVBS produced $HB_sAg$ in the monkey cell producing large T antigen (COS cell).

• Asian-Australasian Journal of Animal Sciences
• /
• v.31 no.2
• /
• pp.167-179
• /
• 2018

Objective: The aim of this study was to create a set of microsatellite markers with high polymorphism for the genetic monitoring and genetic structure analysis of local goose populations. Methods: Novel microsatellite markers were isolated from the genomic DNA of white Roman geese using short tandem repeated probes. The DNA segments, including short tandem repeats, were tested for their variability among four populations of geese from the Changhua Animal Propagation Station (CAPS). The selected microsatellite markers could then be used to monitor genetic variability and study the genetic structures of geese from local geese farms. Results: 14 novel microsatellite loci were isolated. In addition to seven known loci, two multiplex sets were constructed for the detection of genetic variations in geese populations. The average of allele number, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the polymorphism information content were 11.09, 5.145, 0.499, 0.745, and 0.705, respectively. The results of analysis of molecular variance and principal component analysis indicated a contracting white Roman cluster and a spreading Chinese cluster. In white Roman populations, the CAPS populations were depleted to roughly two clusters when K was set equal to 6 in the Bayesian cluster analysis. The founders of private farm populations had a similar genetic structure. Among the Chinese geese populations, the CAPS populations and private populations represented different clads of the phylogenetic tree and individuals from the private populations had uneven genetic characteristics according to various analyses. Conclusion: Based on this study's analyses, we suggest that the CAPS should institute a proper breeding strategy for white Roman geese to avoid further clustering. In addition, for preservation and stable quality, the Chinese geese in the CAPS and the aforementioned proper breeding scheme should be introduced to geese breeders.

• Journal of fish pathology
• /
• v.8 no.1
• /
• pp.37-46
• /
• 1995

The amino acid analysis of polyhedrin protein and nucleotide sequence of polyhedrin gene in H. cunea nuclear polyhedrosis virus (HcNPV) genome have been studied. Polyhedrin had three polypeptide bands in SDS - polyactylamide gel electrophoresis. The major polypeptide had a molecular weight of 25 kd. The polyhedrin was composed of 17 different amino acids. HcNPV DNA was digested with EcoRI restriction enzyme and hybridized with ($\alpha^{32}P$) -labelled AcNPV polyhedrin gene cDNA. The polyhedrin gene was located on the fragment of EcoRI-H. The EcoRI - H fragment containing polyhedrin gene was cloned into the EcoRI site of pUC8 vector which was confirmed with southern blotting, and the recombinant plasmid containg polyhedrin gene was designated as hPE-H. The promoter region of polyhedrin genomic DNA was sequenced. The sequences identified as the TATA box was found at the 5' flanking region of the polyhedrin genomic DNA approximately -79 bp upstream from the transcriptional start site. But CAAT-like box was not shown near the TATA-like box in the polyhedrin gene. Four tandem repeats with the sequence 5' -CTAATAT-3' and 5'-TAAATAA-3' were found between -141 and -108 or -83 upstream and -52 bp downstream from the translation start site. About -141 bp region upstream from the translational start site was highly AT (78%) rich. The coding region for the polyhedrin starts and ends with ATG and TAA, respectively.

• Molecules and Cells
• /
• v.26 no.2
• /
• pp.131-139
• /
• 2008

Expression of the seven open reading frames (ORFs) of single-stranded DNA Curtoviruses such as Beet curly top virus (BCTV) and Beet severe curly top virus (BSCTV) is driven by a bi-directional promoter. To investigate this bidirectional promoter activity with respect to viral late gene expression, transgenic Arabidopsis plants expressing a GUS reporter gene under the control of either the BCTV or BSCTV bi-directional promoter were constructed. Transgenic plants harboring constructs showed higher expression levels when the promoter of the less virulent BCTV was used than when the promoter of the more virulent BSCTV was used. In transgenic seedlings, the reporter gene constructs were expressed primarily in actively dividing tissues such as root tips and apical meristems. As the transgenic plants matured, reporter gene expression diminished but viral infection of mature transgenic plants restored reporter gene expression, particularly in transgenic plants containing BCTV virion-sense gene promoter constructs. A 30 base pair conserved late element (CLE) motif was identified that was present three times in tandem in the BCTV promoter and once in that of BSCTV. Progressive deletion of these repeats from the BCTV promoter resulted in decreased reporter gene expression, but BSCTV promoters in which one or two extra copies of this motif were inserted did not exhibit increased late gene promoter activity. These results demonstrate that Curtovirus late gene expression by virion-sense promoters depends on the developmental stage of the host plant as well as on the number of CLE motifs present in the promoter.

• Korean Journal of Microbiology
• /
• v.40 no.2
• /
• pp.65-72
• /
• 2004

Instability of some eukaryotic sequence propagated in prokaryotic hosts is a frequently observed phenomenon. It is well documented that long inverted repeats, AT-rich sequences with structures like Z-DNA are extremely unstable in E. coli. These sequences may either be under-represented or even lost when cloned in E. coli. When we analyzed the polymorphic pattern for several tandom repeat (TR) in human SCKI gene, we found some TR regions were frequently deleted from plasmids and had difficult problem for their sequencing. These regions may result in non-clonability of the DNA sequence. Here we have cloned two difficult TR regions under low temperature and made two library for DNA sequencing using a nebulizer or sonicator. This study will help to determine the unstable genomic elements in complex mammalian genome.