• Bulletin of the Korean Chemical Society
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• 제34권4호
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• pp.1035-1038
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• 2013

Rolling circle amplification (RCA) of DNA on an aligned-carbon nanotube (a-CNT) surface was electrically interfaced by the a-CNT based filed effect transistor (FET). Since the electric conductance of the a-CNT will be dependent upon its local electric environment, the electric conductance of the FET is expected to give a very distinctive signature of the surface reaction along with this isothermal DNA amplification of the RCA. The a-CNT was initially grown on the quartz wafer with the patterned catalyst by chemical vapor deposition and transferred onto a flexible substrate after the formation of electrodes. After immobilization of a primer DNA, the rolling circle amplification was induced on chip with the a-CNT based FET device. The electric conductance showed a quite rapid increase at the early stage of the surface reaction and then the rate of increase was attenuated to reach a saturated stage of conductance change. It took about an hour to get the conductance saturation from the start of the conductance change. Atomic force microscopy was used as a complementary tool to support the successful amplification of DNA on the device surface. We hope that our results contribute to the efforts in the realization of a reliable nanodevice-based measurement of biologically or clinically important molecules.

• Journal of Microbiology and Biotechnology
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• 제22권5호
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• pp.607-613
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• 2012

Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM $MnCl_2$. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.

• 미생물학회지
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• 제44권2호
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• pp.164-167
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• 2008

PCR 방법과 더불어 최근 핵산증폭기법으로 주목받기 시작한 방법이 Rolling Circle Amplification (RCA) 기법을 응용한 것이다. 본 논문에서 RCA 기술을 활용하는 실험에 이용할 수 있는 특정 길이(N-mer)를 가진 primer의 후보 리스트를 vector 서열이나 특정 미생물 염색체 서열에서 검색할 수 있는 프로그램인 RCA-mer를 개발하였다. RCA-mer는 사용자의 염기서열을 입력으로 받아 이 서열을 대상으로 특정 길이의 N-mer에 대한 존재 유무에 대한 리스트의 후브를 웹으로 보여주는 기능을 가지고 있다. 또한 서로 다른 두 개의 염기서열들에 대해 N-mer가 공통으로 존재하는지, 한쪽 서열에만 존재하는지 확인할 수 있는 기능을 가지도록 개발하였다. 사용자는 선발된 primer 후보들로 부터 적절한 N-mer 서열을 선택하여 실제 RCA를 이용한 실험 곧바로 응용할 수 있도록 후보 primer를 선별하여 사용할 수 있도록 하였다.

• Journal of Microbiology and Biotechnology
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• 제30권5호
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• pp.662-667
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• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• The Plant Pathology Journal
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• 제30권3호
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• pp.310-315
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• 2014

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, is responsible for one of the most devastating viral diseases in tomato-growing countries and is becoming a serious problem in many subtropical and tropical countries. The climate in Korea is getting warmer and developing subtropical features in response to global warming. These changes are being accompanied by TYLCV, which is now becoming a large problem in the Korean tomato industry. The most effective way to reduce damage caused by TYLCV is to breed resistant varieties of tomatoes. To accomplish this, it is necessary to establish a simple inoculation technique for the efficient evaluation of resistance to TYLCV. Here, we present the rolling circle amplification (RCA) method, which employs a bacteriophage using phi-29 DNA polymerase for construction of infectious TYLCV clones. The RCA method is simple, does not require sequence information for cloning, and is less expensive and time consuming than conventional PCR based-methods. Furthermore, RCA-based construction of an infectious clone can be very useful to other emerging and unknown geminiviruses in Korea.

• Genomics & Informatics
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• 제10권4호
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• pp.249-255
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• 2012

In this study, fosmid cloning strategies were used to assess the microbial populations in water from the International Space Station (ISS) drinking water system (henceforth referred to as Prebiocide and Tank A water samples). The goals of this study were: to compare the sensitivity of the fosmid cloning strategy with that of traditional culture-based and 16S rRNA-based approaches and to detect the widest possible spectrum of microbial populations during the water purification process. Initially, microbes could not be cultivated, and conventional PCR failed to amplify 16S rDNA fragments from these low biomass samples. Therefore, randomly primed rolling-circle amplification was used to amplify any DNA that might be present in the samples, followed by size selection by using pulsed-field gel electrophoresis. The amplified high-molecular- weight DNA from both samples was cloned into fosmid vectors. Several hundred clones were randomly selected for sequencing, followed by Blastn/Blastx searches. Sequences encoding specific genes from Burkholderia, a species abundant in the soil and groundwater, were found in both samples. Bradyrhizobium and Mesorhizobium, which belong to rhizobia, a large community of nitrogen fixers often found in association with plant roots, were present in the Prebiocide samples. Ralstonia, which is prevalent in soils with a high heavy metal content, was detected in the Tank A samples. The detection of many unidentified sequences suggests the presence of potentially novel microbial fingerprints. The bacterial diversity detected in this pilot study using a fosmid vector approach was higher than that detected by conventional 16S rRNA gene sequencing.

• 드라이브 ㆍ 컨트롤
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• 제16권4호
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• pp.80-86
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• 2019

The gerotor type device is mainly used in low-speed, high-torque hydraulic motors, and is also applied as a small priming hydraulic pump. For this reason, many studies have been conducted to increase the efficiency of the gerotor pump. In this paper, we propose a new tooth profile design method different from the existing method. The new tooth design is made by modifying the tooth surface using the amplification function of the trajectories, created along the inner and outer rolling circles around the base circle. The shape of the mate rotor is then created using rotation simulation techniques. Such shapes are described as hypercloid. The designed hypercloid rotor is compared with the existing trochoid rotor, and the characteristics of the parameters and volumetric displacements are analyzed. Through this process, the optimum design with larger volumetric displacement than the existing rotor is achieved.

• 미생물학회지
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• 제43권4호
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• pp.243-249
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• 2007

Centromere는 채세포분열과 생식세포분열 등 맡은 주요 기능을 담당하는 고도로 분화된 구조이다. Alphoid DNA (${\alpha}$-satellite)는 인간뿐 아니라 모든 영장류의 염색체 내 centromere에서 발견되는 반복서열의 대부분을 차지한다. 인간 인공염색체(Human Artificial Chromosome, HAC)의 개발에서 가장 핵심적인 부분은 centromere의 분리 및 안정적인 유지에 있다. 이 영역은 출아효모에서 alphoid DNA 반복서열을 hook으로 이용하여 Transformation-associated recombination (TAR) cloning법을 사용하여 선택적으로 분리할 수 있다. 이러한 실험방법으로 먼저 repeat array를 rolling-circle amplication (RCA)를 통하여 약 5 kb까지 길이를 연장시킨 후, 효모내에서 상동성재 조합을 이용한 TAR cloning법을 사용하여 분리할 수 있다. 이렇게 분리된 35 kb-50 kb 길이의 4종류의 centromeric DNA repeat arrays (2,4,5,6 mer)를 사용하여, 반복서열의 안정성 유지를 조사하기 위해 상동성재조 합 변이주인 rad51, rad52, rad54를 사용하여 비교 분석하였다. 야생주, rad51과 rad54 변이주를 이용하여 형질전환을 수행한 결과, 반복서열의 크기에 있어서 많은 변화를 나타내었다. 반면, rad52 변이주는 야생주와 다르게 형질전환빈도가 매우 낮은 비율로 나타났으나, centromeric DNA repeat array의 안정성은 3배 이상으로 높게 나타냈다. 이러한 결과들을 미루어, rad52 변이주를 사용하여 centromeric DNA repeat arrays의 형질전환실험에서 발생하는 맡은 변이를 줄일 수 있을 것으로 보인다. 이러한 유전적 방법은 HAC 제작에서 반복서열의 유지에 훨씬 효율적으로 사용할 수 있을 것으로 사료된다.