• Tuberculosis and Respiratory Diseases
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• v.50 no.3
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• pp.334-342
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• 2001

Background : RpoB gene mutations have been found in about 96-98% of rifampicin (RMP)-resistant Mycobacterium tuberculosis. Recent reports confirm that the in laboratory settings a rpoB gene mutation can be used as a surrogate marker for multi-drug resistant tuberculosis. However, its usefulness in clinical applications has not been evaluated. This study was performed to confirm whether mutation analysis of the rpoB gene of M. tuberculosis is useful in clinical settings. Methods : The medical records of 33 patients in whom rpoB gene analysis was conducted using an INNOLiPA Rif. TB assay (LiPA) from June, 1998, to July, 2000, at the Asan Medical Center were retrospectively reviewed in 33 patients. The clinical characteristics in addition to the drug susceptibility and LiPA results were analyzed. The drug susceptibility test was considered as a gold standard method for M. tuberculosis susceptibility and these results were compared with those of the rpoB gene study and sequencing analysis. Sequencing analysis of the rpoB gene was done in cases where there was a discrepancy between the results of the drug susceptibility an d rpoB gene study. Results : The mean age and sex ratio was $42{\pm}18$, and 24:9 (M:F), respectively. There were 19 RMP susceptible (58%) and 14 RMP-resistant cases (42%) according to the rpoB gene study. The mean time from the request to reporting the results of the rpoB gene study was $5.2{\pm}2.6$ days. The mean gap from reporting the rpoB gene study to reporting the susceptibility was $56{\pm}35$ days. Twenty-eight cases (85%) showed identical results compared with the drug susceptibility results, whereas five cases (15%) showed contradictory results. When compared with the sequencing analysis, of the five cases that showed contradictory results, two had LiP A analysis errors and the remaining three were identical to the sequencing results. The rpoB gene study was of assistance in choosing the appropriate drugs in 28 cases (85%). Conclusions : An rpoB gene study using an LiP A assay was useful in rapidly diagnosing RMP-resistant tuberculosis, which enabled a proper choice of the appropriate drugs in clinical practices. However, an LiPA assay always should be performed in conjunction with microscopy, culture, and susceptibility tests.

• Journal of Crop Science and Biotechnology
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• v.11 no.2
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• pp.91-100
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• 2008

Melting curve analysis of fluorescently labeled DNA fragments is used extensively for genotyping single nucleotide polymorphism(SNP). Here, we evaluated a SNP genotyping method by melting curve analysis with the two probe chemistries in a 384-well plate format on a Roche LightCycler 480. The HybProbe chemistry is based on the fluorescence resonance energy transfer(FRET) and the SimpleProbe chemistry uses a terminal self-quenching fluorophore. We evaluated FRET HybProbes and SimpleProbes for two SNP sites closely linked to two quantitative trait loci(QTL) for southern root-knot nematode resistance. These probes were used to genotype the two parents and 94 $F_2$ plants from the cross of PI 96354$\times$Bossier. The SNP genotypes of all samples determined by the LightCycler software agreed with previously determined SSR genotypes and the SNP genotypes determined on a Luminex 100 flow cytometry instrument. Multiplexed HybProbes for the two SNPs showed a 98.4% success rate and 100% concordance between repeats two of the same 96 DNA samples. Also, we developed a HybProbe assay for the Rcs3 gene conditioning broad resistance to the frogeye leaf spot(FLS) disease. The LightCycler 480 provides rapid PCR on 384-well plate and allows simultaneous amplification and analysis in approximately 2 hours without any additional steps after amplification. This allowed for a reduction of the potential contamination of PCR products, simplicity, and enablement of a streamlined workflow. The melting curve analysis on the LightCycler 480 provided high-throughput and rapid SNP genotyping and appears highly effective for marker-assisted selection in soybean.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.24
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• pp.10647-10652
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• 2015

Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

• Gut and Liver
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• v.12 no.6
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• pp.641-647
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• 2018

Background/Aims: Helicobacter pylori eradication rates are decreasing because of increases in clarithromycin resistance. Thus, finding an easy and accurate method of detecting clarithromycin resistance is important. Methods: We evaluated 70 H. pylori isolates from Korean patients. Dual-labeled peptide nucleic acid (PNA) probes were designed to detect resistance associated with point mutations in 23S ribosomal ribonucleic acid gene domain V (A2142G, A2143G, and T2182C). Data were analyzed by probe-based fluorescence melting curve analysis based on probe-target dissociation temperatures and compared with Sanger sequencing. Results: Among 70 H. pylori isolates, 0, 16, and 58 isolates contained A2142G, A2143G, and T2182C mutations, respectively. PNA probe-based analysis exhibited 100.0% positive predictive values for A2142G and A2143G and a 98.3% positive predictive value for T2182C. PNA probe-based analysis results correlated with 98.6% of Sanger sequencing results (${\kappa}$-value=0.990; standard error, 0.010). Conclusions: H. pylori clarithromycin resistance can be easily and accurately assessed by dual-labeled PNA probe-based melting curve analysis if probes are used based on the appropriate resistance-related mutations. This method is fast, simple, accurate, and adaptable for clinical samples. It may help clinicians choose a precise eradication regimen.

• Fisheries and Aquatic Sciences
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• v.3 no.1
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• pp.64-70
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• 2000

DNA probes for the l6S rRNA have been designed for the detection of Edwardsiella tarda. In order to accomplish this purpose, the l6S rRNA gene from E. tarda has been cloned and sequenced. Two highly feasible oligonucleotide probe sites have been determined by the database analysis programs presented by PCGENE and BLAST. These two probes have been evaluated by slot blot hybridization analysis. Hetero- and homo-trimeric templates have been synthesized using these two probe sites. The templates have been further multimerized by PCR to generate between 150 and 300 bp long DIG-11-dUTP labeled probes. Unlike 3' end labeled oligonucleotide probes or templates, multimerized probes showed no cross­hybridization in the given experimental condition. Furthermore, a significant increase in sensitivity has been observed with these probes. This method, we presented here, may be useful for the designing of probes for the detection of other fish pathogenic microorganisms also.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2004.11a
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• pp.107-116
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• 2004

The advent of microarray technologies gives an opportunity to moni tor the expression of ten thousands of genes, simultaneously. Such microarray data can be deteriorated by experimental errors and image artifacts, which generate non-negligible outliers that are estimated by 15% of typical microarray data. Thus, it is an important issue to detect and correct the se faulty probes prior to high-level data analysis such as classification or clustering. In this paper, we propose a systematic procedure for the detection of faulty probes and its proper correction in Genechip array based on multivariate statistical approaches. Principal component analysis (PCA), one of the most widely used multivariate statistical approaches, has been applied to construct a statistical correlation model with 20 pairs of probes for each gene. And, the faulty probes are identified by inspecting the squared prediction error (SPE) of each probe from the PCA model. Then, the outlying probes are reconstructed by the iterative optimization approach minimizing SPE. We used the public data presented from the gene chip project of human fibroblast cell. Through the application study, the proposed approach showed good performance for probe correction without removing faulty probes, which may be desirable in the viewpoint of the maximum use of data information.

• Biomedical Science Letters
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• v.7 no.1
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• pp.1-5
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• 2001

A cDNA of coagulation Factor IX gene has been screened from the $\lambda$gt11 human fetal liver cDNA library, and used to construct a 2.8-kb full length cDNA after recombining with the N-terminal fragment from pTZ-FIX. Human genomic DNA was isolated, digested with the restriction endonucleases, TaqI, EcoRI, and HindIII, and Southern hybridization was performed using the full length factor IX cDNA as a probe. The hybridized bands generated by the restriction endonucleases were the followings: TaqI, 0.3, 1.0, 1.6, 1.8, 2.7, 3.7, and 5.3 kb bands; EcoRI, 1.8, 4.8, 4.9, 5.5, 6.8, and 12.6 kb bands; HindIII, 4.1, 4.4, 5.2, 5.8, 7.6, and 12.5 kb bands. When the Southern bands were physically mapped along the genome, about 50-kb continuous region harboring almost all of the genomic region of Factor Ⅸ gene was covered. These results suggest a possibility of using an exonal cDNA probe to diagnose abnormalities including large deletions, insertions, and rearrangements along the genome, if there is any.

• Journal of Life Science
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• v.20 no.12
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• pp.1896-1901
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• 2010

This study was conducted using quantitative real-time PCR using Lactobacilli as probiotics. Quantitative real-time PCR (RT PCR) was conducted via a method involving SYBR Green 1 and a probe. Plasmid DNA was cloned using the 16S-23S rRNA intergenic species region. Gene clones were diluted from $10^2$ to $10^{10}$. Standard curves were constructed via Ct values obtained from the results of Real-time PCR via the aforementioned SYBR Green 1 and probe method. Plasmid DNA was also cloned using the 16S-23S rRNA intergenic species region and the gene clones were diluted from $10^2$ to $10^{10}$ copy numbers via the probe method. Using RT PCR, a standard curve of plasmid DNA copy numbers was also determined. The slope value for the Y-axis intercept and $R^2$ value were measured as -3.346, 33.18, and 0.993, respectively, via the first method. For the second method, the slope value for the Y-axis intercept and $R^2$ were -3.321, 31.10 and 0.995, respectively. The PCR inhibitor could not express the detection curve at a copy number over $10^{10}$ via either method, owing to high DNA density. The DNA extract from probiotics was diluted without pre-culturing, and 16 products were amplified via both methods. The Ct value was 11.06~18.12 in the first method and 16.74~22.11 in the second method. Measured probiotics and log copy values were largely similar among the methods used. It was concluded that both methods are effective for analysis, but further research will be required to verify the optimal method.

• Journal of Plant Biology
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• v.35 no.2
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• pp.165-171
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• 1992

In order to study regulation of rbcL gene expression, rbcL gene of chloroplast DNA (Cp DNA) from maize was cloned. Cp DNA was isolated from intact chloroplast and digested with BamHI. BamHI 9 fragment of Cp DNA containing rbcL gene was ligated to pUC19 and transformed into E. coli DH5a. This recombinant plasmid was named pRLYSl. pRLYSl was hybridized with a part of rbcL gene from rice and digested with restriction enzyme BamHI, HindIIl, and PstI. From these results, it was confirmed that pRLYS1 contains intact rbcL gene and orientation of BamHI 9 fragment of Cp DNA in pRLYS1 was determined.rmined.

• Proceedings of the Botanical Society of Korea Conference
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• 1996.07a
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• pp.10-18
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• 1996

Recently, we have reported a rice MADS-box gene, OsMADS1, as a molecular factor triggering flower formation; this has been well studied in a heterologous system (Chung et al., 1994). In order to study whether the OsMADS1 homolog exists in other plant species, the OsMADS1 cDNA was used as a probe to screen a tobacco cDNA library, and a potential homolog, NtMADS3, was isolated. Sequence analysis revealed that the gene shares 56.1% identity in whole amino acids with OsMADS1. Like OsMADS1, the NtMADS3 gene starts to express at a very early stage of flower development, and the expression continues up to flower maturation. In the tobacco flower, the gene is expressed in whorl 2,3 and 4, corresponding to the petal, stamen, and carpel, respectively. Upon ectopic expression in the homologous system, NtMADS3 caused a trasition from inflorescence shoot meristem into floral meristem, reducing flowering time dramatically. These phenotypes strongly suggest the NtMADS3 gene is the OsMADS1 homolog of tobacco. Hybrids between the OsMADS1 and the NtMADS3 plants were also generated. The hybrids flowered even earlier than these two transgenic plants. The detailed studies are discussed here.