• Journal of Microbiology and Biotechnology
• /
• v.30 no.5
• /
• pp.662-667
• /
• 2020

Epidermal growth factor receptor (EGFR) mutations are not only genetic markers for diagnosis but also biomarkers of clinical-response against tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). Among the EGFR mutations, the in-frame deletion mutation in EGFR exon 19 kinase domain (EGFR exon 19-del) is the most frequent mutation, accounting for about 45% of EGFR mutations in NSCLCs. Development of sensitive method for detecting the EGFR mutation is highly required to make a better screening for drug-response in the treatment of NSCLC patients. Here, we developed a fluorometric tandem gene amplification assay for sensitive detection of low-abundance EGFR exon 19-del mutant genomic DNA. The method consists of pre-amplification with PCR, thermal cycling of ligation by Taq ligase, and subsequent rolling circle amplification (RCA). PCR-amplified DNA from genomic DNA samples was used as splint DNA to conjugate both ends of linear padlock DNA, generating circular padlock DNA template for RCA. Long stretches of ssDNA harboring multiple copies of G-quadruplex structure was generated in RCA and detected by thioflavin T (ThT) fluorescence, which is specifically intercalated into the G-quadruplex, emitting strong fluorescence. Sensitivity of tandem gene amplification assay for detection of the EGFR exon 19-del from gDNA was as low as 3.6 pg, and mutant gDNA present in the pooled normal plasma was readily detected as low as 1% fraction. Hence, fluorometric detection of low-abundance EGFR exon 19 deletion mutation using tandem gene amplification may be applicable to clinical diagnosis of NSCLC patients with appropriate TKI treatment.

• Asian Pacific Journal of Cancer Prevention
• /
• v.15 no.18
• /
• pp.7885-7889
• /
• 2014

Background: Activating mutations of epidermal growth factor receptor (EGFR) could predict response to tyrosine kinase inhibitor (TKI) treatment in patients with non-small cell lung cancer (NSCLC). However, the detection of EGFR mutation is frequently challenging in clinical practice for the lack of tumor tissue. The aim of this study was to investigate the feasibility of performing EGFR mutation testing on various types of liquid-based cytology (LBC) samples. Materials and Methods: A total of 434 liquid-based cytology samples were collected from March 2010 and November 2013. Among them, 101 with diagnosis of lung adenocarcinoma had paired surgically resected specimens. The ADx Amplification Refractory Mutation System (ADx-ARMS) was used to determine EGFR mutation status both in LBC and resected samples. Results: All liquid-based cytology samples were adequate for EGFR mutation analysis. The mutation rate was 50.5% in the 434 NSCLC patients with LBC samples and the incidence rates of EGFR mutation were consistent among different specimens. We also detected EGFR positives in 52.5% (53/101) patients with paired histologic specimens. The concordance rate of EGFR mutation between LBC samples and paired histologic specimens was 92.1%. Conclusions: Our results suggest that liquid-based cytology samples are highly reliable for EGFR mutation testing in patients with NSCLC.

• Journal of Microbiology and Biotechnology
• /
• v.23 no.8
• /
• pp.1154-1158
• /
• 2013

Ganciclovir resistance of human cytomegalovirus is associated with mutations in the viral UL97 gene and poses severe problems for immunocompromised patients. In this study, PCR-based restriction fragment length polymorphism and sequencing analyses detected the UL97 D605E mutation in all five clinical isolates from patients with ganciclovir-resistant human cytomegalovirus infection during prolonged ganciclovir therapy, whereas the M460V mutation was only present in 1 of 5 isolates. On the other hand, the detection rates of the D605E mutation in the stored available DNA samples from the donor and allogeneic stem cell transplantation recipients were 66.7% and 93.7%, respectively, suggesting that the presence of D605E mutation was not associated with the ganciclovir exposure. Although the D605E mutation may not be related to ganciclovir resistance, we suggest that this mutation could be an important molecular marker of human cytomegalovirus evolution in East Asian countries. Moreover, the restriction fragment length polymorphism method using the restriction enzyme HaeIII, which is generally used to detect the UL97 A591V mutation, could also detect the D605E mutation and may therefore be a useful tool for future research on the investigation of UL97 gene mutations.

• BMB Reports
• /
• v.46 no.5
• /
• pp.270-275
• /
• 2013

It is essential to analyze rare mutations in many fields of biomedical research. However, the detection of rare mutations is usually failed due to the interference of predominant wild-type DNA surrounded. Herein we describe a sensitive and facile method of detecting rare point mutation on the basis of allele-specific amplification in emulsion PCR. The identification and selective amplification of rare mutation are accomplished in one-pot reaction. The allele-specific primers coupled on magnetic beads allow the exclusive amplification and enrichment of the mutant amplicons. The productive beads bearing mutant amplicons are subsequently stained with the fluorescent dyes. Thus, the rare point mutations with a percentage as low as 0.1%, can be detected by fluorescent analysis. The relative percentages of mutation among different samples can be roughly accessed by counting the fraction of fluorescent positive beads through flow cytometry.

• Bulletin of the Korean Chemical Society
• /
• v.27 no.9
• /
• pp.1346-1352
• /
• 2006

We describe an effective method of microchip electrophoresis (ME) based on single strand conformation poly-morphism (SSCP) analysis to rapidly detect the point mutation, Leu72Met, in a human obesity gene. The 207-bp dsDNA in the Leu72Met region, an estimate of the child obesity DNA mutant, was amplified by polymerase chain reaction (PCR) and submitted to a conventional glass microchip analysis with a sieving matrix of 1.75% poly(vinylpyrrolidone) (Mr 1 300 000), 1.0% poly(ethyleneoxide) (Mr 600 000) and 5.0% w/w glycerol. When combined with base stacking (BS) with hydroxide ions, the SSCP-ME provided rapid analysis as well as sensitive detection. The detection sensitivity was effectively enhanced in the OH- concentration range of 0.01-0.025 M NaOH. The sensitivity and speed of this ME-based SSCP methodology for the rapid detection of Leu72Met point mutations makes this an attractive method for diagnosing childhood obesity in a clinical diagnostic laboratory.

• Journal of KIISE:Computing Practices and Letters
• /
• v.16 no.5
• /
• pp.571-575
• /
• 2010

Mutation testing is known as a very useful technique for measuring the effectiveness of a test data set and finding weak points of the test set. An equivalent mutant degrades the effectiveness of mutation testing. Elimination of equivalent mutants is a very important problem in mutation testing.In this paper, we proposed kinds of methods for detecting class-level equivalent mutants. These methods judge the equivalency of mutants through structural informations and behavioral information of the original program and mutants using static analysis. We found that our approach can detect not a few of equivalent mutants and expected that the cost of mutation testing can be saved considerably.

• Parasites, Hosts and Diseases
• /
• v.59 no.1
• /
• pp.15-22
• /
• 2021

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3' end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56℃ for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

• Journal of Genetic Medicine
• /
• v.16 no.1
• /
• pp.23-26
• /
• 2019

Thrombophilia refers to inherited or acquired hemostatic disorders that result in a predisposition to blood clot formation. When combined with the hypercoagulable state that is characteristic of pregnancy, there is an increased risk of severe and recurrent pregnancy complications. Activated protein C resistance caused by factor V Leiden (FVL) mutation is known to be the most common cause of inherited thrombophilia in Caucasian population. FVL mutation has been related to pregnancy complications associated with hypercoagulation, e.g. miscarriage, intrauterine fetal demise, placental abruption, and intrauterine growth retardation. Although the FVL mutation is easily detected using molecular DNA techniques, patients who are heterozygous for this disorder often remain asymptomatic until they develop a concurrent prothrombotic condition. Because there are potentially serious effects of FVL mutation for pregnancy, and because effective treatment strategies exist, early detection and treatment of this condition might be considered.

• Pediatric Gastroenterology, Hepatology & Nutrition
• /
• v.20 no.1
• /
• pp.65-70
• /
• 2017

Bannayan-Riley-Ruvalcaba syndrome (BRRS) is one of the phosphatase and tensin homolog hamartoma tumor syndrome with a PTEN gene mutation. It is a rare dominant autosomal disorder characterized by cutaneous lipomas, macrocephaly, intestinal polyps, and developmental delay. Diagnosing this syndrome is important, because it may represent the pediatric phenotype of Cowden syndrome, in which there is an increased risk for malignant tumors in children. Until now, the prevalence of BRRS is unknown. Several dozen cases have been reported in the medical literature, but no case has been reported in Korea. Here we report a case of a 19-year-old girl who was diagnosed with BRRS because of macrocephaly, intellectual disability, and intestinal polyps. Her mother had similar findings and a PTEN mutation. Neither patient had mutations detected by conventional mutation-detection techniques, but a PTEN gene deletion was demonstrated by chromosomal microarray analysis.

• The Journal of the Korea Contents Association
• /
• v.7 no.11
• /
• pp.94-101
• /
• 2007

We introduce the way to detect the mutation worm. We implemented the program that can generate signature using LCSeq(Longest Common Subsequence) technique in Suffix Tree studied as pattern recognition algorithm. We also showed the process to detect the mutation of CodeRed worm and Nimda worm and evaluated signatures generated by snort and LCSeq.