• 농업과학연구
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• 제44권3호
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• pp.318-324
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• 2017

With the development of next-generation sequencing (NGS), a cutting-edge technology, genotype-by-sequencing (GBS) became available at a low cost per sample. GBS makes it possible to customize the process of library preparation to obtain high-quality single nucleotide polymorphisms (SNPs) in the most efficient way. However, a GBS library is hard to construct due to fine-tuning of concentration of each reagent and set-up. The major reason for this is the presence of undigested genomic DNA (gDNA) owing to the efficiency of different restriction enzymes for different species with unknown reasons. Therefore, this proof-concept study is to demonstrate the unpredictable patterns of enzyme digestion from various plants in order to make the reader aware of the caution needed when choosing restriction enzymes for their GBS library preparations. Indeed, no pattern was found for the digestibility of gDNA samples and restriction enzymes in the current study. We suggest that more data should be accumulated on this matter to help researchers who want to apply GBS technologies in a variety of genetic approaches.

• 한국정밀공학회:학술대회논문집
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• 한국정밀공학회 2005년도 춘계학술대회 논문집
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• pp.1206-1209
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• 2005

In recent, injection molding process for features in sub-micron scale is under active development as patterning nano-scale features, which can provide the master or stamp for molding, and becomes available around the world. Injection molding has been one of the most efficient processes for mass production of the plastic product, and this process is already applied to nano-technology products successfully such as optical storage media like DVD or BD which is a large area plastic thin substrate with nano-scale features on its surface. Bio chip for like DNA sequencing may be another application of this plastic substrate. The DNA can be sequenced using order of 100 nm pore structure when making the DNA flow through the pore structure. Agarose gel and silicon based chip have been used to sequence the DNA, but injection molded plastic chip may have benefit in terms of cost. This plastic DNA sequencing chip has plenty of pillars in order of 100 nm in diameter on the substrate. When the usual features in case of DVD or BD have very low aspect ratio, even less than 0.5, but the DNA chip will have relatively high aspect ratio of about 2. It is not easy to injection mold the large area thin substrate with sub-micron features on its surface due to the characteristics of the molding process and it becomes much more difficult when the aspect ratio of the features becomes high. We investigated the effect of the molding parameters for injection molding with high aspect ratio nano-scale features and injection molded some plastic DNA sequencing chips. We also fabricated PR masters and Ni stamps of the DNA chip to be used for molding

• 미생물학회지
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• 제38권3호
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• pp.186-191
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• 2002

장내바이러스(enterovirus),로타바이러스(rotavirus),그리고 아데노바이러스(adenovirus)등 물을 통해 전파되는 환경바이러스의 신속한 검출 및 1 차적인 분류를 위해 올리고뉴클레오티드 DNA chip을 이용한 분석 시스템의 유용성에 대하여 연구하였다. BGM 세포배양실험에서 바이러스성 세포병변효과(cytopathic effect) 양성으로 판정된 세포단층으로부터 접종배양 3 일 후 세포내 모든 RNA를 분리하여 중합효소연쇄반응과 DNA chip으로 검출여부 및 유전형을 비교 분석한 결과 세포배양에서 양성으로 판정된 10개의 시료 중 3개가 바이러스 음성으로, 7개가 바이러스 양성으로 나타났다. 중합효소연쇄반응과 DNA chip에 의해 양성으로 나타난 7개 시료의 유전형은 두 방법에 의해 모두 장내바이러스로 동정되어 DNA chip에 의한 1차 동정의 유용성도 증명하였다. 세포배양 후 전기영동분석 없이 일차적인 동정과정까지 불과 3∼4 시간 이내에 수행해낼 수 있는 DNA chip분석은 특이성, 신속성, 및 경제성을 고루 갖추어 환경바이러스 검출방법론의 새로운 영역을 구성할 것으로 사료된다.

• 대한임상검사과학회지
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• 제47권1호
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• pp.17-23
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• 2015

A fine needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant in thyroid nodules. However, between 10 and 30% of the FNABs of thyroid nodules are diagnosed as 'indeterminate'. A molecular method is needed to reduce unnecessary surgery in this group. In Korea, most thyroid cancer is classic papillary type and BRAFV600E mutation is highly prevalent. Thus, this study compared the pyrosequencing method with the conventional direct DNA sequencing and PCR-RFLP analysis and investigated the evaluation of preoperative BRAFV600E mutation analysis as an adjunct diagnostic method with routine FNABs. Sixty-five (78.3%) of 83 histopathologically diagnosed malignant nodule revealed positive BRAFV600E mutation on pyrosequencing analysis. In detail, 65 (83.8%) of 78 papillary thyroid carcinomas sample showed positive BRAFV600E mutation. None of 29 benign nodules had in pyrodequencing, direct DNA sequencing and PCR-RFLP. Out of 31 thyroid nodules classified as 'indeterminate' on cytological examination preoperatively, 28 cases turned out to be malignant: 24 papillary thyroid carcinomas. Among that, 16 (66.7%) classic papillary thyroid carcinomas had BRAFV600E mutation. Among 65 papillary thyroid carcinomas with positive BRAFV600E mutation detected by pyrosequencing analysis, each 3 cases and 5 cases did not show BRAFV600E mutation by direct DNA sequencing and PCR-RFLP analysis. Therefore, pyrosequencing was superior to direct DNA sequencing and PCR-RFLP in detecting the BRAFV600E mutation of thyroid nodules (p =0.027). Detecting BRAFV600E mutation by pyrosequencing was more sensitivity, faster than direct DNA sequencing or PCR-RFLP.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7935-7941
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• 2015

The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage ($>25{\times}$) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.

• Journal of Microbiology
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• 제34권1호
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• pp.25-29
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• 1996

Since sequencing of randomly selected cDNA clones has been known to be a powerful approach to obtain information on gene expression pattern in specific cells or tissues, we have analyzed a 3'-directed cDNA library of vegetative mycelia of A. nidulans by single-pass sequencing of hundreds of randomly selected clones. Sequencing of 292 cDNA clones yielded 209 gene signatures (GSs) probably representing highly or lesser expressed genes in the vegetative mycelia. Among the 209 GSs, 25 (79 cDNA clones) appeared more than once and 184 only once. One GS appeared at a highest frequency of 6 times, 2 GSs5 times, 4 GSs 4 times, a GSs 3 times and 16 GSs twice. About 6.6% GSs comprizing of 13 GSs showed alternative polyadenylation. Among 23 redundant GSs, three were common in both mycelia and sexual organs, and 22 were probably mycelia-specific. Out of 209 GSs, 36 were identified in GenBank showing of 70% or greater similaritis. Only six GSs were for A. nidulans genes, and 13 GSs were of DNA or genes encoding cytoplasmic or organellar proteins. This pattern is similar to those in the human HepG2 cell line and in human colonic mucosa, although very few genes for nuclear proteins and for protein synthesis were in A. nidulans.

• 생명과학회지
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• 제2권2호
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• pp.108-119
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• 1992

근년에 개발된 효소적인 DNA증폭법을 이용하면 일차구조상의 단편적인 정보만 알면 단 수시간내 해석에 필요한 양의 DNA가 증폭되어 cDNA의 염기배열결정의 신속화, 간편화가 가능하게 되었다. 그러므로 유전자증폭법으로써 PCR법에 관해 기술한다. 그리고 리보좀 RNA는 분자시계로서 생물의 계통을 논하는 데에 있어서는 최적의 조건을 갖춘 고분자화합물이다. 이에 PCR법을 이용한 16S like 리보좀DNA의 증폭법을 다루고, PCR증폭산물의 염기배열결정법에 대해 서술한다. 또한 인위적인 leading error 등을 배제하고 신속한 자동해독과 시간적인 절약이 자동 DNA sequencer의 개발과 시판으로 가능하게 되어 cDNA의 형광색소표식 염기배열결정법에 대해서도 서술한다.

• 대한임상검사과학회지
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• 제37권3호
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• pp.173-177
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• 2005

YMDD motif mutants of the hepatitis B virus(HBV) emerge in chronic hepatitis B patients after prolonged lamivudine treatment. HBV DNA breakthrough may be accompained by the emergence of YMDD mutants. We compared the performance of the sequencing method with that of RFMP method in chronic hepatitis B patients who had suffered the HBV DNA breakthrough after lamivudine treatment. Both sequencing and RFMP methods were used to detect YMDD variants in 20 chronic hepatitis B patients. YMDD mutants were detected in 17 samples (85.0%) by both methods. Among them, no mutants were detected in two samples(10.0%), while they were detected in the other sample(5.0%) with the RFMP method. The concordance rate between both methods was 95.0%. There was inconsistency in one sample showing mutants detected by the RFMP method, but not by sequencing method. In the sequencing method, the mutants was detected in the major type virus, but not in the minor type virus. However, both sequencing and RFMP methods were highly concordant except in one sample, so it is suggested that both methods are useful to detect YMDD mutants of chronic hepatitis B patients.

• 대한본초학회지
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• 제28권6호
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• pp.9-14
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• 2013

Objectives : Cnidii Fructus is prescribed as the fruit of Cnidium monnieri (L.) Cusson or Torilis japonica (Houtt.) DC. in Korea pharmacopoeia. Although there are differences in the composition of useful components, two species have been used without distinction. In order to discriminate them, DNA sequencing and taste pattern analysis were used in this study. Methods : Primers ITS 1 and ITS 4 were used to amplify the intergenic transcribed spacer(ITS) region of nuclear ribosomal DNA from seven T. japonica and six C. monnieri samples. Taste pattern of samples were measured by using taste-sensing system SA402B equipped with five foodstuff sensors(CT0, C00, AAE, CA0, and AE1). The five initial taste(sourness, bitterness, astringency, umami, and saltiness) and three aftertaste(aftertaste of bitterness, astringency, and umami) of two species were compared. Results : According to the results of ITS region sequence analysis, two species showed 94 base pairs differences. The similarity of two sequences was 85%. From the taste pattern analysis, sourness, bitterness, aftertaste of bitterness(aftertaste-B), and umami showed a different pattern. Especially, bitterness and aftertaste-B of C. monnieri were significantly higher than T. japonica. In addition, two species were shown to have two markedly different clustering by these two flavors. Conclusion : T. japonica and C. monnieri were effectively discriminated using DNA sequencing and taste pattern analysis. These methods can be used to identify the origin of traditional medicine in order to maintain therapeutic efficacy.

• 대한임상검사과학회지
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• 제47권1호
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• pp.1-5
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• 2015

The nontuberculous mycobacteria (NTM) have been found in different environmental sources. They tend to colonize different body surfaces and secretions. The purpose of this study is to evaluate the presence of NTM in the theater environment. Fifty of Theater environment sample were examined using acid-fast stain, Lowenstein-Jensen medium culture, PCR and DNA-Sequencing. 4 of 50 samples were detected as NFB in AFB stain, L-J medium culture, PCR. and then, All of 4 NTM stains identified as Mycobacterium fortitum type in DNA-sequencing result.