• BMB Reports
• /
• 제37권2호
• /
• pp.148-152
• /
• 2004

A new molecular-baiting method was studied by retrieving targeted gene fragments from an oligonucleotide microarray bait after hybridization. To make the microarray bait, 70-mer oligonucleotides that were designed to specifically represent the SSA1 gene of Saccharomyces cerevisiae were printed on the slide. Samples of the Saccharomyces cerevisiae mRNA were extracted and labeled by the RD-PCR (Restriction Display PCR) method using the Cy5-labelled universal primer, then applied for hybridization. The sample fragments that hybridized to the microarray were stripped, and the eluted cDNAs were retrieved and cloned into the pMD 18-T vector for transformation, plasmid preparation, and sequencing. BLAST searching of the GenBank database identified the retrieved fragments as being identical to the SSA1 gene (from 2057-2541bp). A new method is being established that can retrieve the sample fragments using an oligo-microarray-bait.

• BMB Reports
• /
• 제37권3호
• /
• pp.356-361
• /
• 2004

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.743-743
• /
• 2005

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2005년도 생물공학의 동향(XVII)
• /
• pp.848-848
• /
• 2005

• Mycobiology
• /
• 제46권4호
• /
• pp.448-451
• /
• 2018

Fungal perylenequinones have photodynamic activity and are promising photosensitizers for photodynamic therapy (PDT). Here, we investigated the bactericidal and antitumor activities of phleichrome from the fungal perylenequinone family in vitro. Photodynamic bactericidal activity of phleichrome was analyzed by agar-well diffusion method under dark and illuminated conditions. The photodynamic antitumor activity of phleichrome was analyzed in MCF-7, HeLa, SW480, and HepG2 human cancer cell lines using in vitro cytotoxicity assays. Photodynamic bactericidal activities against Gram-negative and Gram-positive bacteria were species-specific. Antitumor activity against all tumor cell lines increased under the illuminated condition. Depending on the results of the analyses, Phleichrome has potential for further drug development related to its antibacterial and antitumor activities.

• ALGAE
• /
• 제21권2호
• /
• pp.261-266
• /
• 2006

The innate soluble polysaccharides and phenolic compounds of marine macroalgae are serious contaminants which interfere with experimental procedures such as restriction enzyme digestion, polymerase chain reaction (PCR) and other enzymatic reactions using extracted DNA samples. The viscous polysaccharides are co-precipitated with DNA samples by isopropanol or ethanol precipitation in conventional experiment. To overcome the problem, a method for the isolation of high molecular weight DNA from Porphyra tenera is developed with the application of diatomaceous earth column. The isolated DNAs by this method were about 50-100 kb in size and could be digested well with restriction enzymes. The nuclease activity seemed to be minimal, and high reproducibility in the arbitrary primed PCR for RAPD analyses was a distinctive feature. These results suggest that this method is very efficient in isolating nucleic acid from macroalgae including Porphyra.

• Asian-Australasian Journal of Animal Sciences
• /
• 제18권11호
• /
• pp.1529-1534
• /
• 2005

Individual-breed assignments were implemented in six swine populations using twenty six microsatellites recommended by the Food and Agriculture Organization and the International Society for Animal Genetics (FAO-ISAG). Most microsatellites exhibited high polymorphisms as shown by the number of alleles and the polymorphism information content. The assignment accuracy per locus obtained by using the Bayesian method ranged from 33.33% (CGA) to 68.47% (S0068), and the accumulated assignment accuracy of the top ten loci combination added up to 96.40%. The assignment power of microsatellites based on the Bayesian method had positive correlations with the number of alleles and the gene differential coefficient ($G_{st}$) per locus, while it has no relationship to genetic heterozygosity, polymorphism information content per locus and the exclusion probabilities under case II and case III. The percentage of corrected assignment was highest for the Bayesian method, followed by the gene frequency and distancebased methods. The assignment efficiency of microsatellites rose with increase in the number of loci used, and it can reach 98% when using a ten-locus combination. This indicated that such a set of ten microsatellites is sufficient for breed verification purposes.

• Asian-Australasian Journal of Animal Sciences
• /
• 제16권7호
• /
• pp.1066-1070
• /
• 2003

In order to identify differentially expressed mRNAs (which represent possible candidates for significant phenotypic variances of muscle growth, meat quality between introduced European and Chinese indigenous pigs) in the longissimus dorsi muscle tissue between adult Duroc and Erhualian pigs, mRNA differential display was performed. Five 3' anchor primers in combination with 20 different 5' arbitrary primers (100 primer sets) were used and nearly 5,000 cDNA bands were examined, among which 10 differential display cDNAs were obtained, cloned and sequenced. Six of the 10 cDNAs showed similarity to identified genes from GenBank and the other 4 had no matches in GenBank. Differential expression was tested by Northern blot hybridization and could be confirmed for 2 cDNAs. The method used in this study provides a useful molecular tool to investigate genetic variation that occurs at the transcriptional level between different breeds.

• Asian-Australasian Journal of Animal Sciences
• /
• 제20권9호
• /
• pp.1342-1348
• /
• 2007

POU1F1 is a positive regulator for GH, PRL and TSH${\beta}$ and its mutations associate with production traits in ruminant animals. We described a DdeI PCR-RFLP method for detecting a silent allele in the goat POU1F1 gene: TCT (241Ser)>TCG (241Ser). Frequencies of $D_1$ allele varied from 0.600 to 1.000 in Chinese 801 goats. Significant associations of DdeI polymorphism with production traits were found in milk yield (*p<0.05), litter size (*p<0.05) and one-year-old weight (*p<0.05) between different genotypes. Individuals with genotype $D_1D_1$ had a superior performances when compared to those with genotype $D_1D_2$ (*p<0.05). Hence, the POU1F1 gene was suggested to the potential candidate gene for superior milk performance, reproduction trait and weight trait. Genotype $D_1D_1$, characterized by a DdeI PCR-RFLP detection, was recommended to geneticists and breeders as a molecular marker for better performance in the goat industry.

• BMB Reports
• /
• 제40권1호
• /
• pp.82-87
• /
• 2007

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.