• Journal of Genetic Medicine
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• 제12권2호
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• pp.109-117
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• 2015

Purpose: Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make their exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mitochondrial DNA (mtDNA) mutations in 2 Korean families with myoclonic epilepsy with ragged-red fibers (MERRF) and Leigh syndrome, respectively. Materials and Methods: Whole mtDNAs were sequenced by the method of mtDNA-targeted next-generation sequencing (NGS). Results: Two causative mtDNA mutations were identified from the NGS data. An m.8344A>G mutation in the tRNA-Lys gene (MT-TK) was detected in a MERRF patient (family ID: MT132), and an m.9176T>C (p.Leu217Pro) mutation in the mitochondrial ATP6 gene (MT-ATP6) was detected in a Leigh syndrome patient (family ID: MT130). Both mutations, which have been reported several times before in affected individuals, were not found in the control samples. Conclusion: This study suggests that mtDNA-targeted NGS will be helpful for the molecular diagnosis of genetically heterogeneous mitochondrial diseases with complex phenotypes.

• 생명과학회지
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• 제22권11호
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• pp.1587-1594
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• 2012

3C는 진핵세포의 핵에서 크로마틴의 입체 구조/구성을 알아보는 연구 기법이다. 이 기법은 살아있는 세포를 포름알데히드로 처리하여 단백질들 사이의 결합 및 단백질과 DNA 사이의 결합을 고정시킨 후, 제한효소로 DNA를 절단하고, 그 절편들의 연결 빈도를 측정함으로써 DNA 절편 사이의 물리적 근접성을 보여준다. 이 기법을 이용하여 복합 유전자 좌위인 ${\beta}$-글로빈 좌위에서 locus control region이 전사가 활발한 유전자와 가까이 위치하고 있음이 밝혀졌으며, 이러한 결과는 크로마틴 입체 구조가 유전자 전사 조절에 관여함을 나타낸다. 또한 3C 기법은 ChIP 및 genome-wide sequencing과 결합되어 다양한 기술로 진화되었다. 본 총설은 3C의 원리 및 과정을 짚어보고, 3C 기법으로 밝혀진 ${\beta}$-글로빈 좌위의 크로마틴 입체 구조를 설명하고자 하며, 나아가 3C를 기본으로 한 다양한 응용 연구 기법도 살펴보고자 한다.

• Molecules and Cells
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• 제44권8호
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• pp.602-612
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• 2021

DNA methylation is an important epigenetic mechanism affecting genome structure, gene regulation, and the silencing of transposable elements. Cell- and tissue-specific methylation patterns are critical for differentiation and development in eukaryotes. Dynamic spatiotemporal methylation data in these cells or tissues is, therefore, of great interest. However, the construction of bisulfite sequencing libraries can be challenging if the starting material is limited or the genome size is small, such as in Arabidopsis. Here, we describe detailed methods for the purification of Arabidopsis embryos at all stages, and the construction of comprehensive bisulfite libraries from small quantities of input. We constructed bisulfite libraries by releasing embryos from intact seeds, using a different approach for each developmental stage, and manually picking single-embryo with microcapillaries. From these libraries, reliable Arabidopsis methylome data were collected allowing, on average, 11-fold coverage of the genome using as few as five globular, heart, and torpedo embryos as raw input material without the need for DNA purification step. On the other hand, purified DNA from as few as eight bending torpedo embryos or a single mature embryo is sufficient for library construction when RNase A is treated before DNA extraction. This method can be broadly applied to cells from different tissues or cells from other model organisms. Methylome construction can be achieved using a minimal amount of input material using our method; thereby, it has the potential to increase our understanding of dynamic spatiotemporal methylation patterns in model organisms.

• Plant Breeding and Biotechnology
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• 제5권4호
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• pp.269-281
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• 2017

With the continual development of genetically modified (GM) crops, it has become necessary to develop detailed and effective molecular characterization methods to select candidate events from a large pool of transformation events. Relative to traditional molecular analysis methods such as the polymerase chain reaction (PCR) and Southern blot hybridization, next generation sequencing (NGS) technology for whole-genome sequencing of complex crop genomes had proven comparatively useful for in-depth molecular characterization. In this study, four transformation events, including one in Bacillus thuringiensis (Bt)-resistant rice, one in resveratrol-producing rice, and two in beta-carotene-enhanced soybeans, were selected for molecular characterization. To merge NGS analysis and Southern blot-hybridization results, we confirmed the transgene insertion sites, insertion construction, and insertion numbers of these four transformation events. In addition, the read-coverage depth assessed by NGS analysis for inserted genes might provide consistent results in terms of inserted T-DNA numbers in case of complex insertion structures and highly duplicated donor genomes; however, PCR-based methods can produce incorrect conclusions. Our combined method provides an effective and complete analytical approach for whole-genome visual inspection of transformation events that require biosafety assessment.

• Pediatric Infection and Vaccine
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• 제6권1호
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• pp.123-130
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• 1999

목 적 : 무균성뇌막염은 소아에서 주로 발생하는 질환으로, 흔히 장 바이러스에 의해 초래 된다. 저자들은 그동안 우리나라에서 분리된 무균성뇌막염 원인바이러스의 5'-NCR에 대한 sequencing을 통하여 prototype과의 homology를 비교하고 이 연구를 진단에 이용하기 위한 기초자료로 이용하기 위하여 본 연구를 시도하였다. 방 법 : 과거 4년간 우리나라에서 분리한 장바이러스 Coxsackie B1, Echovirus 3, 7, 9, 30 을 이용하여 RNA를 분리하고 RT-PCR을 이용하여 DNA를 합성한 후, direct sequencing을 이용하여 WHO에서 얻은 prototype과 homology를 비교하였다. 결 과 : 1) PCR product는 155bp와 440bp부위에 특징적인 띠를 관찰할 수 있었다. 2) 155bp에 관한 sequencing homology를 보면 prototype의 Coxsackie virus와 echovirus 는 92.1%의 homology를 보였다. 3) 환자에서 분리한 Coxsackie B1은 prototype과 94.1%의 homology를 보였다. 4) 환자에서 분리한 Echovirus 3은 92.8%, Echovirus 7은 92.8%, Echovirus 9는 94.1%, Echovirus 30은 82.9%의 homology를 보였다. 결 론 : 장바이러스의 5'-NCR은 homology가 높아서 진단에 이용하기에 좋으며 이 부위를 통한 typing을 위해서는 더 긴 부위를 sequencing할 필요가 있다.

• Genomics & Informatics
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• 제12권2호
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• pp.50-57
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• 2014

We present a new next-generation sequencing-based method to identify somatic mutations of lung cancer. It is a comprehensive mutation profiling protocol to detect somatic mutations in 30 genes found frequently in lung adenocarcinoma. The total length of the target regions is 107 kb, and a capture assay was designed to cover 99% of it. This method exhibited about 97% mean coverage at $30{\times}$ sequencing depth and 42% average specificity when sequencing of more than 3.25 Gb was carried out for the normal sample. We discovered 513 variations from targeted exome sequencing of lung cancer cells, which is 3.9-fold higher than in the normal sample. The variations in cancer cells included previously reported somatic mutations in the COSMIC database, such as variations in TP53, KRAS, and STK11 of sample H-23 and in EGFR of sample H-1650, especially with more than $1,000{\times}$ coverage. Among the somatic mutations, up to 91% of single nucleotide polymorphisms from the two cancer samples were validated by DNA microarray-based genotyping. Our results demonstrated the feasibility of high-throughput mutation profiling with lung adenocarcinoma samples, and the profiling method can be used as a robust and effective protocol for somatic variant screening.

• 대한임상검사과학회지
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• 제38권2호
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• pp.99-104
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• 2006

파필로마바이러스(Human papilloma virus; HPV)는 자궁경부암의 주요한 원인균으로 30종 이상의 여성성기감염과 관련된 유전자형이 보고되었으며 자궁경부암과 관련성이 높은 고위험군과 관련성이 낮은 저위험군으로 나뉘어 진다. 최근 HPV 유전자형의 임상적 활용이 높아짐에 따라 신속하고 정확하게 HPV 유전자형을 선별할 수 있는 방법이 요구되고 있다. 본 연구의 목적은 여러 가지 분자생물학적 방법 중에서 정확도가 높은 DNA 염기서열분석을 이용하여 한국인 여성에서 HPV의 유전자형분포와 빈도를 구하고자 하였다. 전국 각 지역의 3,978명으로부터 채취한 자궁경부 검체에서 DNA를 추출하고, HPV L1 유전자 영역에서 PCR을 실시하였다. PCR 양성이 나온 경우 DNA 염기서열분석을 실시하였으며 GenBank BLAST program을 이용하여 HPV 유전자형을 분석하였다. 검사대상의 평균 년령은 37.6세였으며 년령 범위는 20-73세였고, 30대 여성이 검사를 가장 많이 실시하였다(42.2%). 총 3.978명 중에서 1,174명(1,174/3,978, 29.5%)이 HPV 양성을 보였으며 136명(11.6%)이 중복감염을 보여, 총 1,310개의 HPV 유전자를 분석하였다. 본 연구에서는 21종의 고위험군, 16종의 저위험군을 포함하여 총 37종의 HPV 유전자형이 검출되었으며, HPV 고위험군의 빈도는 69.8%(914/1,310), 저위험군은 26.0% (340/1,310)로 나타났다. 년령은 20대에서 HPV 양성률이 가장 낮았으며(69.5%), 60대 이상의 검체에서 발견된 HPV는 대부분이 고위험군이었다. 고위험군에서는 HPV 16형이 13.21%로 가장 높게 나타났으며, HPV 53형이 9.62%, 58형이 9.24%로 높게 나타났다. 다음으로 HPV 70(5.50%), 33(4.73%), 66(4.20%), 18(4.05%), 52 (4.05%), 31(3.97%), 56(3.51%)의 순으로 나타났다. 저위험군에서는 HPV 62(4.20%), 61(3.89%), 6(3.59%), 81(3.59%), 84(3.51%), 11(2.6%)의 순으로 검출되었다. DNA 염기서열분석을 이용한 한국인 여성의 HPV 유전자형빈도 분석 결과는 HPV의 역학적 연구와 백신개발을 위한 자료로 유용할 것이며, 자궁경부암의 치료와 관련한 특이적 HPV 유전자형 관련 연구에 도움을 줄 것으로 사료된다.

• Biotechnology and Bioprocess Engineering:BBE
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• 제8권2호
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• pp.83-88
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• 2003

A cellulose-producing strain isolated from rotten apples was identified as Gluconacetobacter hansenii based on its physiological properties and the 16S rDNA complete sequencing method, and specifically named Gluconacetobacter hansenii PJK. The amount of bacterial cellulose (BC) produced by G. hansenii PJK in a shaking incubator was 1.5 times higher than that produced in a static culture. The addition of ethanol to the medium during cultivation enhanced the productivity of bacterial cellulose, plus the supplementation of 1% ethanol into the culture medium made the produced BC aggregate into a big lump and thus protected the bacterial-cellulose-producing G. hansenii PJK cells in the shear stress field from being converted into non-cellulose-producing (Cel) mutants. Cells subcultured three times in a medium containing ethanol retained their ability to produce BC without any loss in the production yield.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2001년도 제2회 생물정보학 국제심포지엄
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• pp.11-35
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• 2001

For the last twenty years fragment assembly in DNA sequencing followed the "overlap - layout - consensus"paradigm that is used in all currently available assembly tools. Although this approach proved to be useful in assembling clones, it faces difficulties in genomic shotgun assembly: the existing algorithms make assembly errors and are often unable to resolve repeats even in prokaryotic genomes. Biologists are well-aware of these errors and are forced to carry additional experiments to verify the assembled contigs. We abandon the classical “overlap - layout - consensus”approach in favor of a new Eulerian Superpath approach that, for the first time, resolves the problem of repeats in fragment assembly. Our main result is the reduction of the fragment assembly to a variation of the classical Eulerian path problem. This reduction opens new possibilities for repeat resolution and allows one to generate error-free solutions of the large-scale fragment assemble problems. The major improvement of EULER over other algorithms is that it resolves all repeats except long perfect repeats that are theoretically impossible to resolve without additional experiments.

• 미생물학회지
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• 제24권2호
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• pp.79-85
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• 1986

A. nidulans의 $tRNA^{Arg}$의 염기순서를 효소절단 방법으로 결정하였다. 이 방법으로 염기순서를 결정한 결과 다음과 같았다. 5'GGCCGGCUGGCCCAAXUGGCAAGGCXUCUGAXUACGAAXCAGGAGAUUGCAXXXXXGAGCXXUXXGUCGGUCACCA3'. 위의 결과로 플로버잎 구조를 만들어본 결과 안티코돈이 ACG인 $tRNA^{Arg}$으로 판명되었고. 이 결과는 아미노산 부하검사(charging test)의 결과와 일치하였다. 이 tRNA의 유천자의 염기순서 결과와 비교하여 염기순서의 정확성을 검증하였고, minor base분석을 통하여 전 염기순서를 추정하였다.