• Bulletin of the Korean Chemical Society
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• v.34 no.4
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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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• v.22 no.5
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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.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.164-167
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• 2008

Recently, rolling circle amplification (RCA) technique has been widely focused in the field of gene amplification just like PCR method. We have developed RCA-mer, which is a web-based program searching for primer candidates from a given sequence. It can be applied to find primer lists in DNA amplification experiment based on RCA method. The RCA-mer compares 8-mer primer lists with user's input sequence such as vector, mitochondria, and microbial genome sequence. After calculating 8-mers existences in a given sequence, it displays matched and no-matched primer lists with their GC-contents. In addition to it, RCA-mer can search the existence of 8-mer primer lists in two sequences whether they are co-existed or not. Users can apply candidate primer lists to their researches which use RCA techniques.

• Journal of Microbiology and Biotechnology
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• v.30 no.5
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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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• v.30 no.3
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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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• v.10 no.4
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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.

• Journal of Drive and Control
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• v.16 no.4
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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.

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