• Analytical Science and Technology
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• v.31 no.2
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• pp.71-77
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• 2018

Detection of bloodstains is a very important process in scientific investigations, and luminol is often used for the detection of bloodstains that are not visible. Recently, new preparation methods of blood detection reagents based on luminol (BloodFlareA, B) were developed and reported to have higher active persistence and to be more economical than conventional blood detection reagent, BlueStar forensic. In this paper, we tested the specificity and effect of the BloodFlares (A and B) on DNA and compared them with those of BlueStar forensic. False positive results for the BloodFlares were not observed in semen, saliva, vaginal fluids, urine, sweat, and nasal discharge, but were observed in $CuSO_4$, $FeSO_4$, and bleach solutions, and the observed patterns were similar to those of BlueStar forensic. The effect on DNA was determined by analyzing the DNA yield, degradation index, and DNA profiling. Based on these results, we concluded that the BloodFlares based on luminol do not affect DNA stability and are applicable in forensics.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.63-66
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• 1995

Soybean nuclear extracts were prepared to detect SEF3(soybean embryo factor 3), which is presumed to be a trans-acting factor for the expression of the soybean ${\beta}-conglycinin\;{\alpha}'$ subunit gene. To increase the specific activity of DNA probe during labeling with $[{\alpha}-^{32}P]$dATP, dATP was added to a final concentration of 1.1 mM during the chase reaction. It results in approximately four-fold increase of specific activity of the DNA probe. Effects of several modifications in preparation of soybean nuclear extracts were examined. It was found that glycerol is effective to stabilize SEF3 during the preparation of nuclear extracts and polyethylenimine could be used to increase the specific activity of SEF3 in nuclear extracts.

• Bulletin of the Korean Chemical Society
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• v.31 no.2
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• pp.435-441
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• 2010

We report the preparation and characterization of a new and water soluble complex of palladium(II) with 1,10- phenanthroline and butyldithiocarbamate ligands. This compound has been studied through spectroscopic techniques, $^1H$ NMR, IR, electronic spectra and elemental analysis and conductivity measurements. The complex shows 50% cytotoxic concentration ($Ic_{50}$) value against chronic myelogenous leukemia cell line, K562, much lower than that of cisplatin. Thus the mode of binding of this complex to calf thymus DNA have been extensively investigated by isothermal titration UV-visible spectrophotometry, fluorescence, gel filteration and other methods. UV-visible studies show that the complex exhibits cooperative binding with DNA and remarkably denatures the DNA at extremely low concentration ($~13\;{\mu}M$). Fluorescence studies indicate that the complex intercalate into DNA. Gel filtration studies suggest that the binding of Pd(II) complex with DNA is strong enough that it does not readily break. In these interaction studies, several thermodynamic and binding parameters are also determined which may reflect the mechanism of action of this type of compound with DNA.

• Journal of Pharmaceutical Investigation
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• v.33 no.3
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• pp.209-213
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• 2003

The objective of this study was to construct the pegylated liposomes containing plasmid DNA with optimal encapsulation efficiency. Plasmid DNA $(pGL2\;clone\;753,\;{\sim}6\;kb)$ was encapsulated by the freeze/thawing method into liposomes composed of 1-palmitoyl-2-oleyl-sn-glycerol-3-phosphocholine (POPC), didodecyl dimethyl ammonium bromide (DDAB), distearoylphosphatidyl ethanolamine polyethylene glycol 2000 (DSPE-PEG 2000) and DSPE-PEG 2000-male-imide. The liposomes containing plasmid DNA were then extruded through two stacked polycarbonate filters with series of different pore sizes to control the liposome size. The plasmid DNA entrapped in the liposomes was separated from free plasmid DNA by Sephadex CL-4B column chromatography. The decreased pore size of polycarbonate filters resulted in the decreased size of liposomes. The encapsulation efficiency was markedly affected by the cationic lipid (DDAB) concentration, but to a low degree by the size of liposomes and by the amount of plasmid DNA.

• Journal of Pharmaceutical Investigation
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• v.37 no.1
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• pp.39-43
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• 2007

Previously, we have reported that PLGA nanoparticles were prepared for sustained release of water-soluble blue dextran and the particle size, in vitro release pattern and encapsulation were modulated by varying polymers. This study was designed to encapsulate plasmid DNA in PLGA nanoparticles and to investigate the effect of Polymers and temperatures. PLGA nanoparticles were fabricated with poloxamer 188 (P188) or poloxamer 407 (P407) by using spontaneous emulsification solvent diffusion method. As a model plasmid DNA, pCMV-Taq2B/1L-18 was encapsulated in PLGA nanoparticles. Then, the particle size, zeta potential and encapsulation efficiency of nanoparticles containing plasmid DNA were investigated. Particle sizes of PLGA nanoparticles prepared with P188 and P407 were in the range of 200-330 nm and 250-290 nm, respectively. Zeta potentials of nanoparticles were negative regardless of nanoparticle compositions. Encapsulation efficiency of P407 nanoparticles prepared at $30^{\circ}C$ was higher than those at other preparation condition. From the results, the PLGA nanoparticles prepared with poloxamers at different temperature, could modulate the particles size of nanoparticles, and encapsulation efficiency of plasmid DNA.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1455-1459
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• 2014

We report a sensitive and reliable FRET-based nanotechnology assay for efficient detection and quantification of bisulfite-unmodified or modified DNA. Bisulfite-untreated DNA or bisulfite-treated DNA is subjected to PCR amplification with biotin-conjugated primers so that the amounts of bisulfite-untreated and treated DNA can be differentiated. Streptavidin-coated quantum dots (QDs) are used to capture biotinylated PCR products intercalated with SYBR Green, enabling FRET measurement. Key features of our method include its low intrinsic background noise, high resolution, and high sensitivity, enabling detection of as little as 1.75 ng of bisulfite-untreated DNA in the presence of an approximately 1,000-fold excess of bisulfite-untreated DNA compared to bisulfate-treated DNA, with the use of PCR reduced (as low as 15 cycles). SYBR Green as an intercalating dye as well as a FRET acceptor allows for a single-step preparation without the need for primers or probes to be chemically conjugated to an organic fluorophore. Multiple acceptors per FRET donor significantly enhance the signal-to-noise ratio as well. In consideration of the high relevance of bisulfite treatment to DNA methylation quantitation, our system for FRET measurement between QDs and intercalating dyes can be generally utilized to analyze DNA methylation and to potentially benefit the scientific and clinical community.

• Molecular & Cellular Toxicology
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• v.4 no.3
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• pp.246-252
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• 2008

Tumor tissue is usually contaminated by normal tissue components, which reduces the sensitivity of analysis for exploring genetic alterations. Although microdissection has been adopted to minimize the contamination of tumor DNA with normal cell components, there is a concern over the amount of microdissected DNA not enough to be applied to array-CGH reaction. To amplify the extracted DNA, several whole genome amplification (WGA) methods have been developed, but objective comparison of the array-CGH outputs using different types of WGA methods is still scarce. In this study, we compared the performance of non-amplified microdissected DNA and DNA amplified in 2 WGA methods such as degenerative oligonucleotide primed (DOP)-PCR, and multiple strand displacement amplification (MDA) using Phi 29 DNA polymerase. Genomic DNA was also used to make a comparison. We applied those 4 DNAs to whole genome BAC array to compare the false positive detection rate (FPDR) and sensitivity in detecting copy number alterations under the same hybridization condition. As a result microdissected DNA method showed the lowest FPDR and the highest sensitivity. Among WGA methods, DOP-PCR amplified DNA showed better sensitivity but similar FPDR to MDA-amplified method. These results demonstrate the advantage and applicability of microdissection for array-CGH analysis, and provide useful information for choosing amplification methods to study copy number alterations, especially based on precancerous and microscopically invaded lesions.

• Applied Chemistry for Engineering
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• v.18 no.6
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• pp.607-611
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• 2007

To improve transfection efficiency, we prepared histidine-low molecular weight water-soluble chitosan (LMWSC) having the potential to form complex with DNA as a cationic polymer. Histidine-LMWSC was synthesized by the esterification reaction and removing phthaloyl group. The histidine-LMWSC was characterized using FT-IR, $^1H$ NMR spectra. Histidine-LMWSC was complexed with plasmid DNA (pDNA) in various polymer/DNA (N/P) weight ratios, and the complex was identified using gel retardation assay. The particle sizes of the hisitidine-LMWSC/DNA complexes were measured on a DLS instrument by fixing the histidine-LMWSC/DNA weight ratio of 10/1. Owing to the utilization of a large excess amount of cationic LMWSC against anionic DNA, the particle size of histidine-LMWSC/DNA complexes was in the range of 100~200 nm. Therefore, histidine-LMWSC will be useful in the development of gene carriers.

• The Plant Pathology Journal
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• v.25 no.2
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• pp.184-188
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• 2009

Identification of differently expressed genes under specific tissues and/or environments provides insights into the nature and underlying mechanisms of cellular processes. Although cDNA-AFLP (Amplified Fragment Length Polymorphism) is a powerful method for analyzing differentially expressed genes, its use has been limited to the requirement of radioactive isotope use and the difficulty of isolating the bands of interest from a gel. Here, we describe a modified method for cDNA-AFLP that uses a fluorescence dye for detection and isolation of bands directly from a small size polyacrylamide gel. This method involves three steps: (i) preparation of cDNA templates, (ii) PCR amplification and differential display, and (iii) identification of differentially expressed genes. To demonstrate its utility and efficiency, differentially expressed genes during vegetative growth and appressorial development of Magnaporthe oryzae were analyzed. This method could be applied to compare gene expression profiles in a diverse array of organisms.

• Korean Journal of Microbiology
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• v.38 no.2
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• pp.103-106
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• 2002

Direct PCR from intact fungal cells is not readily suitable to all fungi mainly because of difficulties in rupturing the cell walls. Microwave irradiation has been proven to be useful in fungal DNA extraction protocol. Here we describe a fast template preparation method for PCR amplification from Aspefillus nidulans conidiospores using microwave irradiation. We optimized the duration far microwave irradiation, and the amount of template DNA for PCR. Amplification from samples prepared in this manner was so efficient that we could get PCR products with size enough to identify transformants. We believe that this is a time-saving procedure for screening true transformants of A. nidulans.