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

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.

Analytical Performance Evaluation of a Digital Real-Time PCR for Quantifying Major BCR::ABL1 Transcripts.

BACKGROUND: Accurate quantification of the BCR::ABL1 transcripts is essential for measurable residual disease (MRD) monitoring in chronic myeloid leukemia (CML) after tyrosine kinase inhibitor (TKI) treatment. This study evaluated the newly developed digital real-time PCR method, Dr. PCR, as an alternative reverse transcription-PCR (qRT-PCR) for MRD detection. METHODS: The performance of Dr. PCR was assessed using reference and clinical materials. Precision, linearity, and correlation with qRT-PCR were evaluated. MRD levels detected by Dr. PCR were compared with qRT-PCR, and practical advantages were investigated. RESULTS: Dr. PCR detected MRD up to 0.0032%IS (MR4.5) with excellent precision and linearity and showed a strong correlation with qRT-PCR results. Notably, Dr. PCR identified higher levels of MRD in 12.7% (29/229) of patients than qRT-PCR, including six cases of MR4, which is a critical level for TKI discontinuation. Dr. PCR also allowed for sufficient ABL1 copies in all cases, while qRT-PCR necessitated multiple repeat tests in 3.5% (8/229) of cases. CONCLUSION: Our study provides a body of evidence supporting the clinical application of Dr. PCR as a rapid and efficient method for assessing MRD in patients with CML under the current treatment regimen.

Comparison of digital PCR platforms using the molecular marker.

Assays of clinical diagnosis and species identification using molecular markers are performed according to a quantitative method in consideration of sensitivity, cost, speed, convenience, and specificity. However, typical polymerase chain reaction (PCR) assay is difficult to quantify and have various limitations. In addition, to perform quantitative analysis with the quantitative real-time PCR (qRT-PCR) equipment, a standard curve or normalization using reference genes is essential. Within the last a decade, previous studies have reported that the digital PCR (dPCR) assay, a third-generation PCR, can be applied in various fields by overcoming the shortcomings of typical PCR and qRT-PCR assays. We selected Stilla Naica System (Stilla Technologies), Droplet Digital PCR Technology (Bio-Rad), and Lab on an Array Digital Real-Time PCR analyzer system (OPTOLANE) for comparative analysis among the various droplet digital PCR platforms currently in use commercially. Our previous study discovered a molecular marker that can distinguish Hanwoo species (Korean native cattle) using Hanwoo-specific genomic structural variation. Here, we report the pros and cons of the operation of each dPCR platform from various perspectives using this species identification marker. In conclusion, we hope that this study will help researchers to select suitable dPCR platforms according to their purpose and resources.

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

BACKGROUND: We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). METHODS: We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. RESULTS: The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. CONCLUSIONS: PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis.

Detection of genetic variation using dual-labeled peptide nucleic acid (PNA) probe-based melting point analysis.

BACKGROUND: Thermal denaturation of probe-target hybrid is highly reproducible, and which makes probe melting point analysis reliable in the detection of mutations, polymorphisms and epigenetic differences in DNA. To improve resolution of these detections, we used dual-labeled (quencher and fluorescence), full base of peptide nucleic acid (PNA) probe for fluorescence probe based melting point analysis. Because of their uncharged nature and peptide bond-linked backbone, PNA probes have more favorable hybridization properties, which make a large difference in the melting temperature between specific hybridization and partial hybridization. RESULTS: Here, we have shown that full base dual-labeled PNA is apt material for fluorescence probe-based melting point analysis with large difference in the melting temperature between full specific hybridization and that of partial hybridization, including insertion and deletion. In case of narrowly distributed mutations, PNA probe effectively detects three mutations in a single reaction tube with three probes. Moreover, we successfully diagnose virus analogues with amplification and melting temperature signal. Lastly, Melting temperature of PNA oligomer can be easily adjusted just by adding gamma-modified PNA probe. CONCLUSIONS: The PNA probes offer advantage of improved flexibility in probe design, which could be used in various applications in mutation detection among a wide range of spectrums.