• Animal cells and systems
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• v.5 no.2
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• pp.153-156
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• 2001

A key aspect of genomic research in the “post-genome era”is to associate sequence variations with heritable phenotypes. The most common variations in the human genome are single nucleotide polymorphisms (SNPs) that occur approximately once in every 500 to 1,000 bases. Although analyzing the phenotypic outcome of these SNPs is crucial to facilitate large-scale association studies of genetic diseases, detection of SNPs from an extended number of human DNA samples is often difficult, labor-intensive and time-consuming. Recent development in SNP detection methods using DNA microarrays and mass spectrophotometry has allowed automated high throughput analyses, but such equipments are not accessible to many scientists. In this study, we demonstrate that a simple PCR-based method using primers with a mismatched base at the 3'-end provides a fast and easy tool to identify known SNPs from human genomic DNA in a regular molecular biology laboratory. Results from this PCR amplification of specific alleles (PASA) analysis efficiently and accurately typed the Q576R polymorphism of human IL4 receptor from the genomic DNAs of 29 Koreans, including 9 samples whose genotype could not be discerned by the conventiona1 PCR-SSCP (single strand conformation polymorphism) method. Given the increasing attention to disease-associated polymorphisms in genomic research, this alternative technique will be very useful to identify SNPs in large-scale population studies.

• The Plant Pathology Journal
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• v.35 no.1
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• pp.71-76
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• 2019

$SdhB^{P225F}$ and $SdhB^{H272R}$ mutations have been found associated with boscalid resistance in Botrytis cinerea from strawberry in Shanghai, China. For rapid detection of two mutations, tetra-primers were designed and optimized to gain the relatively high accuracy and specificity based on the ARMS-PCR technique, by which resistance can be identified with different lengths of products on agarose gels. The tetra-primer ARMS-PCR systems for $SdhB^{P225F}$ and $SdhB^{H272R}$ were validated by 9 SdhB-squenced strains repeatedly. Then, sensitivity of 30 more strains were also tested by the methods, which were accordant with genotypes by sequencing and the sensitivity of conidial germination to boscalid by 100%. Thus, the methods developed in this study are proved to be rapid, inexpensive, accurate and practical for resistance detection of Botrytis cinerea caused by $SdhB^{P225F}$ and $SdhB^{H272R}$ mutations.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.6
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• pp.891-895
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• 2019

Objective: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the ${\alpha}$-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). Methods: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). Results: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources ($0.25ng/{\mu}L$). Conclusion: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

• Journal of Korean Society of Forest Science
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• v.102 no.2
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• pp.223-228
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• 2013

Phytoplasmas were detected consistently in 42 mulberry cultivars showing dwarf disease using DNA analysis by amplification with phytoplasma universal primer pairs P1/P7 (about 1.8 kb and R16F2n/R2 (about 1.2 kb). The point mutation from 42 cultivars of mulberry tree was detected by single-strand conformation polymorphism (SSCP) analysis. The SSCP profiles were clearly observed from all of cultivars in 8% polyacrylamide gel, electrophoresizing for and running 8-15 hrs. at 150V, $10^{\circ}C$. The MD and JWB phytoplasma PCR products was mixed and electrophoresis was performed to detect their polymorphism. In this results, the SSCP profiles of all bands of MD and JWB were analyzed on single lane and were distinct in their each of band patterns. The SSCP analysis was possible to detect of 1.8 kb and 1.2 kb nucleotide size and near close band patterns were distinct by mix of two samples. Previously, it was only possible to detect of point mutation under 600 bp nucleotide sequence by SSCP analysis but this modification of SSCP technique was possible to detect clearly SSCP band patterns of about 1.8 kb and 1.2 kb nucleotides.

• Journal of Life Science
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• v.29 no.6
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• pp.653-661
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• 2019

The first small interfering RNA (siRNA) therapeutics have recently been approved by the Food and Drug Administration in the U.S., and the demand for a new RNA therapeutics bioanalysis method-which is essential for pharmacokinetics, including the absorption, distribution, metabolism, and excretion of siRNA therapeutics-is rapidly increasing. The stem-loop real-time qPCR (RT-qPCR) assay is a useful molecular technique for the identification and quantification of small RNA (e.g., micro RNA and siRNA) and can be applied for the bioanalysis of siRNA therapeutics. When the anti-HPV E6/E7 siRNA therapeutic was used in preclinical trials, the established stem-loop RT-qPCR assay was validated. The limit of detection was sensitive up to 10 fM and the lower limit of quantification up to 100 fM. In fact, the reliability of the established test method was further validated in three intra assays. Here, the correlation coefficient of $R^2$>0.99, the slope of -3.10 ~ -3.40, and the recovery rate within ${\pm}20%$ of the siRNA standard curve confirm its excellent robustness. Finally, the circulation profiles of siRNAs were demonstrated in rat serum, and the pharmacokinetic properties of the anti-HPV E6/E7 siRNA therapeutic were characterized using a stem-loop RT-qPCR assay. Therefore, the stemloop RT-qPCR assay enables accurate, precise, and sensitive siRNA duplex quantification and is suitable for the quantification of small RNA therapeutics using small volumes of biological samples.

• Journal of the Korean Society of Fisheries and Ocean Technology
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• v.9 no.1
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• pp.19-23
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• 1973

The method of forecasting advection fog at Busan area in July is developed using the Spreen's scatter-diagraam technique. The used Parameters are (1) air temperature (2) dew-point temperature, (3) sea surface temperature (4) resultantt wind direction (5) resultant wind speed in Busan. The skill score and the pcr cent correct based on 4 yeare of dependent data are 0.79 and 90.3% respectively.

• Journal of Life Science
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• v.25 no.8
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• pp.903-909
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• 2015

Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO₄, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.

• Tuberculosis and Respiratory Diseases
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• v.69 no.4
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• pp.271-278
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• 2010

Background: Recent studies have demonstrated that the epidermal growth factor receptor (EGFR) genotype is the most important predictive marker to EGFR-tyrosine kinase inhibitors (TKIs) and first-line gefitinib treatment will be approved in the near future for use in non-small cell lung cancer (NSCLC) patients with the EGFR mutation. Direct sequencing is known to be the standard for detecting EGFR mutations; however, it has limited sensitivity. Peptide nucleic acids (PNA)-mediated PCR clamping method is a newly introduced method for analyzing EGFR mutations with increased sensitivity and stability. Methods: A total of 71 NSCLC patients were analyzed for EGFR mutations using the PNA-mediated PCR clamping technique. Sixty-nine patients were analyzed for clinicopathologic correlation with EGFR genotype; 2 patients with indeterminate results were excluded. In order to determine EGFR-TKI drug response, 57 patients (42 gefitinib, 15 erlotinib) were included in the analysis. Results: The EGFR mutation rate was 47.8%. Being female, a non-smoker, and having adenocarcinoma were favorable clinicopathologic factors, as expected. However, more than a few smokers (33.3%), male (28.1%), and patients with non-adenocarcinoma (28.6%) had the EGFR mutation. Having a combination of favorable clinicopathologic factors did not increase the EGFR mutation rate significantly. Drug response to EGFR-TKIs showed significant differences depending on the EGFR genotype; ORR was 14.3% for wild type vs 69.0% for mutant type; DCR is 28.6% for wild type vs 96.6% for mutant type. The median EGFR-TKI treatment duration is 7.6 months for mutant type group and 1.4 months for wild type group. Conclusion: EGFR genotype determined using the PNA-mediated PCR clamping method is significantly correlated with the clinical EGFR-TKI responses and PNA-mediated PCR.

• Journal of Korean Society of Forest Science
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• v.87 no.3
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• pp.334-340
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• 1998

This research was conducted to identify plant regulatory regions by gene tagging method. A promoterless GUS coding sequence was introduced to Populus nigra ${\times}$ P. maximowiczii via Agrobacterium strains(LBA4404/EHA101), and putative transgenic poplars were selected by culturing on medium containing G418($60mg/{\ell}$) and by GUS assay. Among them one positive plant was to amplify the native sequences flanking to the introduced GUS gene in plant genome by inverse PCR method and from this 730 by DNA product was obtained. After subcloning and sequencing, it has 88% homology to the Eucalyptus gunnii CAD(cinnamyl alcohol dehydrogenase) gene. The GUS gene fused with the putative promoter reinserted into poplar leaves by particle bombardment method to test the funtional promoter activity. Upon staining with X-gluc, many blue spots appeared on the leaf segments bombarded by the chimeric gene 2-3 days, thus the isolated DNA fragment contain some possible coding region as well as a putative regulatory sequences of poplar CAD gene.

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1561-1569
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• 2006

Denaturing gradient gel electrophoresis (DGGE) is one of the most frequently used methods for analysis of soil microbial community structure. Unbiased PCR amplification of target DNA templates is crucial for efficient detection of multiple microbial populations mixed in soil. In this study, DGGE profiles were compared using different pairs of primers targeting different hypervariable regions of thirteen representative soil bacteria and clones. The primer set (1070f-1392r) for the E. coli numbering 1,071-1,391 region could not resolve all the 16S rDNA fragments of the representative bacteria and clones, and moreover, yielded spurious bands in DGGE profiles. For the E. coli numbering 353-514 region, various forward primers were designed to investigate the efficiency of PCR amplification. A degenerate forward primer (F357IW) often yielded multiple bands for a certain single 16S rDNA fragment in DGGE analysis, whereas nondegenerate primers (338f, F338T2, F338I2) differentially amplified each of the fragments in the mixture according to the position and the number of primer-template mismatches. A forward primer (F352T) designed to have one internal mismatch commonly with all the thirteen 16S rDNA fragments efficiently produced and separated all the target DNA bands with similar intensities in the DGGE profiles. This primer set F352T-519r consistently yielded the best DGGE banding profiles when tested with various soil samples. Touchdown PCR intensified the uneven amplification, and lowering the annealing temperature had no significant effect on the DGGE profiles. These results showed that PCR amplification bias could be much improved by properly designing primers for use in fingerprinting soil bacterial communities with the DGGE technique.