High-Resolution Melting (HRM) analysis of DNA methylation using semiconductor chip-based digital PCR

Jeong Jinuk,Yang Yongsu,Song Min-Sik,Won Hee-Young,Han Andrew T.,Kim Songmi 한국유전학회 2024 Genes & Genomics Vol.46 No.8

Background Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. Objective A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. Methods In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. Results Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. Conclusion The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation. Background Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. Objective A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. Methods In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. Results Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. Conclusion The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.

Design and Implementation of Bioluminescence Signal Analysis Tool

Jeong, Hye-Jin,Lee, Byeong-Il,Hwang, Hae-Gil,Song, Soo-Min,Min, Jung-Joon,Choi, Heung-Kook Korea Multimedia Society 2006 멀티미디어학회논문지 Vol.9 No.12

The term molecular imaging can be broadly defined as the in vivo characterization and measurement of biologic processes at the cellular and molecular level. Optical imaging that has highly reproducibility and repetition used in molecular imaging research. In the bioluminescence imaging, animals carrying the luciferase gene are imaged with a cooled CCD(Charge-Coupled Device) camera to pick up the small number of photons transmitted through tissues. Molecular imaging analysis will allow us to observe the incipience and progression of the disease. But hardware device for molecular imaging and software for molecular image analysis were dependent on imports. In this paper, we suggest image processing methods and designed software for bioluminescence signal analysis. And we demonstrated high correlation(r=0.99) between our software's photon counts and commercial software's photon counts. ROI function and processing functions were accomplished without error. This study have the importance of the development software for bioluminescence image processing and analysis. And this study built the foundations for creative development of analysis methods. We expected this study lead the development of image technology.

Computational Modeling of Novel Phosphoinositol-3-kinase γ Inhibitors Using Molecular Docking, Molecular Dynamics, and 3D-QSAR

Suparna Ghosh,Seketoulie Keretsu,Seung Joo Cho 대한화학회 2021 Bulletin of the Korean Chemical Society Vol.42 No.8

Phosphoinositol-3-kinase ? (PI3K?) is a member of the class-IB PI3K superfamily and plays a significant role in G-protein-coupled receptor mediated cell signaling. Recent studies have suggested that elevated expression of PI3K? in tumor-associated macrophages strongly influences immune suppression and tumor growth. Due to the presence of many isoforms of PI3K, the selective inhibition of PI3K? remains challenging. Therefore, it is necessary to design more potent inhibitors against PI3K? for cancer treatment. In this study, we have reported the critical interactions of isoindolinone-based inhibitors with PI3K? by docking and molecular dynamics simulations. The binding free energy of the receptor-ligand complex was calculated using molecular mechanics/Poison-Boltzmann surface area approach. We have performed the comparative molecular field analysis (CoMFA) and the comparative molecular similarity indices analysis (CoMSIA) to determine the structure?activity relationship of the inhibitors. The CoMFA (q2 = 0.681 and r2 = 0.968) and CoMSIA (q2 = 0.665 and r2 = 0.982) models showed reasonable predictive ability. Thereafter, the contour maps derived from CoMFA and CoMSIA were used to design several new compounds, among which, the compound D04 showed high predicted activity values. The designed compound was subjected to absorption-distribution-metabolism-excretion/toxicity prediction and synthetic accessibility analyses. Our results could provide theoretical guidance for the future development of new PI3K? inhibitors.

자폐장애 환자에서 FMR-1 유전 삼염기 반복의 분자생물학적 분석

곽호순,정철호,전효진,장은진,김희철,김정범,박영남 대한소아청소년정신의학회 2000 소아청소년정신의학 Vol.11 No.1

연구목적 : 자폐장애의 원인을 유전학적으로 규명하려는 연구가 시도되고 있으며, 그 중 fragile-X 증후군과의 연관성에 대한 연구가 활발히 진행되고 있다. fragile-X 염색체(Xq27.3)는 세포유전학적 방법으로 증명할 수 있으나 검사에 많은 제약과 단점이 있으므로, 본 연구에서는 보다 신뢰성이 높은 분자 생물학적 방법으로 FMR-1 유전자내 CGG 삼염기 반복부위를 분석하여 자폐장애와 fragile-X 증후군의 연관성을 규명하고자 하였다. 방법 : 자폐장애 환아(99명)와 정상대조군(8명)을 대상으로 FMR-1 유전자의 CGG 반복배열 부위를 sense와 antisense primer를 이용하여 PCR법으로 분석하엿으며, 동시에 세포유전학적 검사도 시행하였다. PCR 분석에서 CGG 반복수가 50 이상인 경우에 대해서는 StB12.3 혹은 Pfxa3 probe를 이용한 Sourthern blot hybridization으로 확인하였다. 결과 : FMR-1 유전자의 CGG 반복배열에 대한 PCR 분석 결과 CGG 삼염기의 반복배열의 수는 자폐장애 환자군과 정상대조군 사이에 통계적으로 유의한 차이가 없었다(p=0.207). 자폐장애 환자에서 CGG 반복수가 50회 이상인 조기변이(premutation) 환자가 2명 있었으나 Sourthern blot hybridization 결과 완전변이(full mutation)로 판정할 수 있는 경우는 없었다. 세포유전학적 검사에서 환자군 모두에서 정상 핵형을 나타내었으며 fragile-X 염색체는 확인되지 않았다. 결론 : 이상의 결과에서 자페장애 환자가 FMR-1 유전자의 CGG 삼염기 반복부위 이상, 즉 fragile-X 염색체 이상을 동반하지 않았음을 증명할 수 있었다. 이는 fragile-X 증후군을 자�장애의 직접적인 원인이라고 보기에는 어려움이 있음을 시사한다. Objectives : There has been a rapid expansion of studies aimed at elucidating the genetic basis of autistic disorder, especially it's relationship to fragile-X syndrome. The detection of fragile X chromosome(Xq27.3) by cytogenetic analysis has revealed many difficulties in testing. Therefore, to explore the relationship between autistic disorder and fragile X syndrome, this study administered molecular biologic methods which examined an unstable CGG repeat within the fragile X mental retardation-1(FMR-1) gene. Methods : Ninety nine autistic children and eight normal control children were tested. The number of CGG repeats within FMR-1 gene was measured after amplification by PCR, and cytogenetic analysis was also carried out to detect fragile site Xq27.3. Sourthern blot hybridization, using StB12.3 and/or Pfxa3 probe, was done for the patients showing expansion of more than 50 CGG repeats(premutation). Results : All but two autistic patients had no expansion in CGG repeats by PCR and there was no significant statistical difference in number of CGG repeat in comparison with normal control. Two autistic patients, considered as premutation by PCR analysis, had no full mutation or premutation by Southern blot hybridization. All autistic children tested did not have any abnormal karyotype or fragile site Xq27.3. Conclusions : These results suggest that autistic patients may not have abnormality in FMR-1 gene or abnormal expansion in CGG repeat. In conclusion, fragile X syndrome may not be antecedent of autistic disorder. KEY WORDS : Autistic disorder·FMR-1 gene trinucleotide repeats·Molecular biologic analysis.

A Study on the Trend of Molecular Diagnosis of COVID- 19 through Reviewing of the Review Method

Hae-Ryoung Park ASCONS 2022 IJBSA Vol.4 No.1

Background/Objectives: The purpose of this study was to review the molecular diagnosis of coronavirus disease as COVID-19 was a worldwide pandemic from 2019. In addition, by providing accurate information on the molecular diagnostic tools currently in use, it was intended to be helpful in determining the timing of treatment and in public health policies. Methods/Statistical analysis: In this study, to examine the use of diagnostic tools related to COVID-19, “Omicron”, “Diagnosis”, “RT-PCR”, “COVID-19”, and “Analysis” were searched as keywords through Google and the literature on the latest diagnostic techniques was used. Findings: Recently, it was announced that the fatality rate of Omicron, which has a high epidemic rate and high infection rate, was 1/4 of that of Delta. SARS-CoV-2 Omicron variant RT-PCR laboratory developed assay was said to be sensitive and specific for detecting Omicrons in nasopharyngeal and nasal swabs. The closed tube Penn-RAMP can be used with minimal equipment and training. Improvements/Applications: The purpose of this study was to investigate the types and characteristics of accurate molecular diagnostic tests. These molecular diagnostic tests are considered to be important for prompt and accurate diagnosis, distinguishing infected persons, and coping with appropriate isolation and treatment. In addition, this study was intended to help the general public understand molecular diagnostic testing methods.

Systematic Review and Meta-Analysis of Damage Associated Molecular Patterns HMGB1 and S100B in Schizophrenia

Michael Mackey,Laurena Holleran,Gary Donohoe,Declan P. McKernan 대한신경정신의학회 2022 PSYCHIATRY INVESTIGATION Vol.19 No.12

Objective Immune system dysregulation is hypothesised to be central to the aetiopathogenesis of schizophrenia; however, the role of sterile inflammation remains unclear. Damage associated molecular patterns are key initiators of sterile inflammation and are detectable in peripheral blood. Methods A defined systematic search of the Web of Science, PubMed, and Scopus was performed to identify adult case-control studies published between January 1990 and June 2022. Three studies consisting of 242 cases and 83 controls met inclusion for the systematic review and meta-analysis of HMGB1 while twenty-eight studies consisting of 1,544 cases and 1,248 healthy controls were included for S100B. Results A significant standardised mean difference in peripheral S100B and HMGB1 concentrations was detected between cases and controls. S100B subgroup analysis determined the largest significant effect size for unmedicated individuals diagnosed with schizophrenia. Conclusion This study provides evidence that peripheral S100B and HMGB1 concentrations are elevated in individuals diagnosed with schizophrenia when compared with healthy controls. These results should be interpreted with caution as significant heterogeneity was present during meta-analysis of S100B in the entire sample and in sub-group analysis. The persistence of significant heterogeneity throughout subgroup analysis indicates that the current diagnostic groupings may be a barrier to understanding human behaviours and emotions.

New record of Gayralia kuroshiensis (Ulotrichales: Chlorophyta) in Korea based on morphological and molecular analyses

Prismabella Wilis Andiska,양미연,김명숙 국립중앙과학관 2023 Journal of Asia-Pacific Biodiversity Vol.16 No.4

Monostromatic algae, Monostroma and Gayralia, are taxonomically complex, with several nomenclatural changes over the past years owing to indistinguishable morphological features. Several Monostroma species were recently transferred to the genus Gayralia based on their ontogeny and molecular analysis. There is no record of the occurrence of Gayralia in Korea, whereas three species of Monostroma have been reported. We conducted a molecular analysis based on the nuclear ribosomal internal transcribed spacer region of the specimens from Jeju, Korea, that we considered to be included in the genus Monostroma. The specimens from this study and five sequences of Monostroma nitidum downloaded from the GenBank were conspecific with Gayralia kuroshiensis from Japan but distinct from the M. nitidum syntype from Australia. G. kuroshiensis has a single, expanded, laminar, monostromatic thallus that is light to midꠓgreen. The thallus thickness in the marginal regions was similar to that of G. kuroshiensis from Japan, with an undulate shape and no marginal teeth. The cells in surface view were in groups of two, rectangular to polygonal in shape, with one to three pyrenoids per cell. Based on the morphological and molecular analyses, we report a new record of G. kuroshiensis in Korea.

Molecular modeling and experimental verification of lipase-catalyzed enantioselective esterification of racemic naproxen in supercritical carbon dioxide

권정훈,Jeong Yeong Jeong,강정원 한국화학공학회 2009 Korean Journal of Chemical Engineering Vol.26 No.1

Experimental and simulation analyses were performed on the lipase-catalyzed esterification reaction of racemic naproxen by CALB (candida antarctica lipase B) enzyme in supercritical carbon dioxide. The reaction pathways were investigated by quantum mechanical analysis, and the enantioselectivity of the products was predicted by molecular dynamics simulation analysis. Calculated results from molecular modeling in supercritical carbon dioxide were qualitatively compared with experimental data by using racemic naproxen as a substrate. All molecular modeling results and experimental data were acquired and compared with those in ambient and supercritical condition. Moreover, to verify the stability of enzymatic reaction in each solvent condition, reaction pathways were investigated in several solvent conditions (vacuum, water, hexane and supercritical carbon dioxide), and the stability of enzymatic reaction in supercritical carbon dioxide was compared with other solvent conditions.

Molecular analysis of <i>myocilin</i> and <i>optineurin</i> genes in Korean primary glaucoma patients

Park, Joonhong,Kim, Myungshin,Park, Chan Kee,Chae, Hyojin,Lee, Seungok,Kim, Yonggoo,Jang, Woori,Chi, Hyun Young,Park, Hae-Young Lopilly,Park, Shin Hae D.A. Spandidos 2016 MOLECULAR MEDICINE REPORTS Vol.14 No.3

<P>To investigate the underlying genetic influences of primary glaucoma in Korea, molecular analysis was performed in 112 sporadic cases, and results compared with healthy controls. The <I>myocilin</I> (<I>MYOC</I>) and <I>optineurin</I> (<I>OPTN</I>) genes were directly sequenced in 112 unrelated patients, including 17 with primary open-angle glaucoma, 19 with juvenile open-angle glaucoma, and 76 with normal tension glaucoma. Healthy unrelated Korean individuals (n=100) were used as the non-selected population control. A total of three <I>MYOC</I> and four <I>OPTN</I> variants potentially associated with primary glaucoma were identified in 4 and 18 patients, respectively. A novel variant of <I>MYOC</I>, <I>p.Leu255Pro</I>, was predicted to be potentially pathogenic by <I>in silico</I> analysis. Another, <I>p.Thr353Ile</I>, has been previously reported. These two missense variants were detected in patients with a family history of glaucoma. Combined heterozygous variants <I>p.[Thr123=;Ile288=]</I> were identified in 2 of 112 (2%) patients but not in healthy controls. Among <I>OPTN</I> variants, a novel variant <I>p.Arg271Cys</I> was identified. Homozygous <I>p.[Thr34=;Thr34=]</I> (4/112, 4%), homozygous <I>p.[Met98Lys;Met98Lys]</I> (4/112, 4%), or combined heterozygous <I>p.[Thr34=;Arg545Gln]</I> (9/112, 8%) was significantly associated with the development of primary glaucoma [odds ratio (OR)=8.768, 95% confidence interval (CI)=1.972–38.988; relative risk=1.818, 95% CI=1.473–2.244; P=0.001]. The present study provides insight into the genetic or haplotype variants of <I>MYOC</I> and <I>OPTN</I> genes contributing to primary glaucoma. Haplotype variants identified in the present study may be regarded as potential contributing factors of primary glaucoma in Korea. Further studies, including those on additional genes, are required to elucidate the underlying pathogenic mechanism using a larger cohort to provide additional statistical power.</P>

Analysis of phase transition, structural and dynamical properties of R290 using molecular dynamics simulation

Md. Sarwar Alam,Ji Hwan Jeong 대한기계학회 2020 JOURNAL OF MECHANICAL SCIENCE AND TECHNOLOGY Vol.34 No.10

Propane (R290), a hydrocarbon refrigerant, is an excellent choice of cooling fluids for use in refrigeration and air conditioning systems considering the environmental point of view and system performance. The phase transition phenomenon and structural and dynamic properties of R290 were analyzed through a molecular dynamics (MD) simulation. The densities, isobaric heat capacities and viscosities were computed and the variations of density, volume, potential energy and the nucleation process were examined to investigate the effects of condensation temperature on the phase transition rate. The mean square displacement and velocity autocorrelation function for different temperatures were simulated for dynamical analysis. Radial distribution functions were investigated to get insight into the structural analysis at the atomic level. Shear viscosity and isobaric heat capacity obtained by the present simulation showed a good agreement with the REFPROP data. The structural analysis revealed that the phase transition of R290 did not affect its intramolecular structure.