• Korean Journal Plant Pathology
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• v.12 no.2
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• pp.187-190
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• 1996

백합으로부터 total RNA를 분리하여 LSV 외피단백질 유전자의 551 bp에 해당하는 특정 염기서열을 증폭할 수 있는 primer로 RT-PCR를 수행하였다. 그 결과 Lilium oriental hybrid cvs. Miani, Marco Polo, Casablanca, Le Reve 품종에서 551 bp의 DNA 절편이 증폭되었고 이 절편의 염기서열을 분석한 결과 LSV외피단백질 유전자의 일부임을 확인할 수 있었다. 그러므로 RT-PCR 방법으로, 실험에 사용하 s4품종 모두 LSV에 감염되어 있음을 알 수 있었다.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.806-810
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• 1999

A multiplex-polymerase chain reaction (PCR) assay was used to detect staphylococcal enterotoxin production by 12 strains of Staphylococcus aureus isolated from clinical specimens. To evaluate the efficacy and/or sensitivity of this method, the results were compared to those obtained with the reversed passive latex agglutination kit (SET-RPLA, Denka Seiken, Japan). Of 10 strains positive by PCR were positive by RPLA but two strains, representing high sensitivity of the former method. Enterotoxin B was the most prevalent by the two methods. The kappa index between the two methods was 0.826, indicating a higher agreement and fully reliable for use. These results would suggest that sensitive, inexpensive, and relatively rapid multiplex-PCR technique may be an effective means for the detection of staphylococcal enterotoxin genes as an alternative to traditional methods such as kits or immunological methods, which depend upon the amount of enterotoxin produced.

• Research in Plant Disease
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• v.7 no.3
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• pp.123-133
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• 2001

Many pathovars of the species Pseudomonas syringae are known to produce different phytotoxins as secondary metabolites. Although phytotoxins generally enhance the virulence of P. syringae, they are not required for pathogenesis. Among the phytotoxins produced by P. syringae, lipodepsipeptides, coronatine, phaseolotoxin, and tabtoxin are the most well-known toxins which have been intensively studied for their structure, mode of action, biosynthesis, and regulation. A polymerase chain reaction (PCR) technique that amplifies a segment of the phytotoxin gene cluster using a primer set has been developed in recent years. This method offers the advantages of speed and sensitivity compared to the approaches based on physiological and biochemical methods. PCR detection of genes involved in the production of toxins could be exploited for early diagnosis of plant diseases caused by P. syringae pathovars.

• Korean Journal of Microbiology
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• v.40 no.3
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• pp.254-256
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• 2004

Polymerase chain reaction (PCR) is a powerful tool for precisely amplifying selected DNA sequences that have had a broad impact on genomic studies. When examining human $\alpha$- and $\beta$- tryptase genes which have 95% DNA homology, inconsistent PCR amplification of genomic sequences hampered our progress. This study suggests that long PCR technique on the original DNA digested with restriction enzymes improves both efficiency and sensitivity of PCR. These improved results seem to derived from the effective denaturation of the original genomic DNA template or reduction of formation of secondary structures that block either primer annealing or extension in PCR. Elimination of homo- or hetero-duplex products derived from highly homologous genes provides an additional advantage in this study. This communication describes how the use of restriction enzymes improved these efficiencies, and also facilitated studies of highly homologous genes including tryptase genes.

• Journal of Microbiology
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• v.44 no.2
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• pp.155-161
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• 2006

Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63 % (137 of 217) in the standard PCR method to about 34 % (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: ${\alpha},\;{\beta},\;{\gamma}-$ Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivation-based method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

• Research in Plant Disease
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• v.17 no.2
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• pp.169-176
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• 2011

Phytophthora katsurae is the fungus responsible for chestnut ink disease. The objectives of this study were to determine if a single-strand conformation polymorphism (SSCP) analysis of rDNA-ITS region, elongation factor 1 alpha gene and ${\beta}$-tubulin gene could be used for rapid identification and genetic diversity of P. katsurae, and to assess the potential use of the SSCP technique as a diagnostic tool for P. katsurae. Each regions amplified by PCR using primers designed to overlap the genus Phytophthora were characterized for the Phytophthora species. PCR products were denatured and electrophoresed for SSCP analysis. P. katsurae isolates showed an unique pattern in SSCP analysis and were easily distinguished from other Phytophthora species used as the control. This indicates that SSCP analysis is an useful technique for distinguishing Phytophthora species from genetically close relatives, and show that the SSCP analysis of each region is an efficient detection tool for P. katsurae. But PCR-SSCP analysis of single-gene may have difficulty in distinguishing P. katsurae from other Phytophthora species. Therefore, PCR-SSCP analysis of multi-genes can be useful for rapid and effective identification of P. katsurae.

• Tuberculosis and Respiratory Diseases
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• v.44 no.5
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• pp.1001-1010
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• 1997

Background : PCR technique is useful in diagnosis of pulmonary tuberculosis. But, its sensitivity and specificity is some different among several studies. Our aim is compare our PCR results with other's previous PCR results in AFB smear negative patients. Methods : PCR were performed in patients that their disease were suspected as active pulmonary tuberculosis and that their initial serial sputum AFB smear results were negative. Total number of patients studied by PCR technique was 177. Also, we analyzed the data only in patients whose bronchial washing fluid AFB smear was negative. And the primer had been used was IS 6110. Results : In our retrograde study, the number of patients who are diagnosed as having active pulmonary tuberculosis, inactive pulmonary tuberculosis and nontuberculous pulmonary disease was 99, 28, 50, respectively. In the sputum study, the sensitivity of PCR is 41.5% (27 PCR positive cases/65 active TBc cases). And the sensitivity of TB culture is 53.8% (35 TB culture positive cases/65 active TBc cases). In the bronchial washing specimen study, the sensitivity of PCR is 53.8% (21 PCR positive cases/39 active TBc cases). And the sensitivity of TB culture is 43.6% (17 TB culture Positive cases/39 active TBc cases). The specificity of PCR in our study is 94.9%. (74 PCR negative cases/78 inactive TBc or nontubereulous cases) In the cases of patients who were never takened anti-TBc medication, the sensitivity of PCR (45.6%--25 positive cases/55 cases) is some lower than culture (58.2%--32 positive cases/55 cases). In the cases of patients who had been takened anti-TBC medication. the sensitivity of PCR (60%--18 positive cases/30 cases) is some superior than culture (50%--15 positive cases/30 cases). Conclusion : We think that PCR results in cases of sputum AFB smear negative patients is nearly same as culture. And PCR is especially useful in patients who had been takened anti-TBc medication on admission.

• BMB Reports
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• v.33 no.2
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• pp.162-165
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• 2000

Polymerase chain reaction (PCR) is well established as an indispensable tool of molecular biology; and yet a limitation for cloning applications continues to be that products often require subsequent restriction to be that products often require subsequent restriction digests, blunt-end ligation, or the use of special linear vectors. Here a rapid, PCR-based system is described for the simple, restriction enzyme-free generation of synthetic, 'restriction-like' DNA fragments with staggered ends. Any 3'- or 5'-protruding terminus, but also non-palindromic overhangs with an unrestricted single strand length are specifically created. With longer overhangs, "Restriction-PCR" does not even require a ligation step prior to transformation. Thereby the technique presents a powerful tool e.g. for a successive, authentic reconstitution of sub-fragments of long genes with no need to manipulate the sequence or to introduce restriction sites. Since restriction enzyme-free and thereby devoid the limitations of partial DNA digests, "Restriction-PCR" allows a straight one-step generation and cloning of difficult DNA fragments that internally carry additional sites for specific sequence insertions or deletions can be precisely engineered into genes of interest. With these properties "Restriction-PCR" has the potential to add significant speed and versatility to a wide variety of DNA cloning applications.

• Journal of Genetic Medicine
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• v.12 no.2
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• pp.72-78
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• 2015

Recently, noninvasive prenatal test (NIPT) has been adopted as a primary screening tool for fetal chromosomal aneuploidy. The principle of NIPT lies in isolating the fetal fraction of cell-free DNA in maternal plasma and analyzing it with bioinformatic tools to measure the amount of gene from the target chromosome, such as chromosomes 21, 18, and 13. NIPT will contribute to decreasing the need for unnecessary invasive procedures, including amniocentesis and chorionic villi sampling, for confirming fetal aneuploidy because of its higher positive predictive value than that of the conventional prenatal screening method. However, its greater cost than that of the current antenatal screening protocol may be an obstacle to the adoption of this innovative technique in clinical practice. Digital polymerase chain reaction (dPCR) is a novel approach for detecting and quantifying nucleic acid. dPCR provides real-time diagnostic advantages with higher sensitivity, accuracy, and absolute quantification than conventional quantitative PCR. Since the groundbreaking discovery that fetal cell-free nucleic acid exists in maternal plasma was reported, dPCR has been used for the quantification of fetal DNA and for screening for fetal aneuploidy. It has been suggested that dPCR will decrease the cost by targeting specific sequences in the target chromosome, and dPCR-based noninvasive testing will facilitate progress toward the implementation of a noninvasive approach for screening for trisomy 21, 18, and 13. In this review, we highlight the principle of dPCR and discuss its future implications in clinical practice.

• BMB Reports
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• v.40 no.3
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• pp.444-447
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• 2007

Polymerase chain reaction (PCR) is a powerful technique in molecular biology and is widely used in various fields. By amplifying DNA fragments, PCR has facilitated gene cloning procedures, as well as molecular genotyping. However, the extraction of DNA from samples often acts as a limiting step of these reactions. In particular, the extraction of PCR-compatible genomic DNA from higher plants requires complicated processes and tedious work because plant cells have rigid cell walls and contain various endogenous PCR inhibitors, including polyphenolic compounds. We recently developed a novel solution, referred to as AnyDirect, which can amplify target DNA fragments directly from whole blood without the need for DNA extraction. Here, we developed a simple lysis system that could produce an appropriate template for direct PCR with AnyDirect PCR buffer, making possible the direct amplification of DNA fragments from plant leaves. Thus, our experimental procedure provides a simple, convenient, non-hazardous, inexpensive, and rapid process for the amplification of DNA from plant tissue.