Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform

Department of Hematology, Affiliated People's Hospital of Jiangsu University, Zhenjiang, Jiangsu 212002, PR China.
Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.82). 12/2010; 411(23-24):2097-100. DOI: 10.1016/j.cca.2010.08.020
Source: PubMed


Detection of the JAK2 mutation has recently been included under the essential diagnostic criteria for myeloproliferative neoplasm (MPN). High-resolution melt (HRM) curve analysis, a nongel-based, automated system, is introduced as a means of mutation scanning without the requirement of any post-PCR handling.
We studied the sensitivity and reproducibility of LightScanner™ platform in the detection of JAK2 V617F mutation and the availability for diagnostic use in MPN.
The reproducible sensitivity of HRM analysis with LightScanner™ platform was 5% for the detection of JAK2 V617F mutation. In the test of blind screening of 105 samples (48 Ph- MPN and 57 Ph+ chronic myeloid leukemia), the identical judgement was interpreted by two blinded investigators. HRM analysis of all cases was fully concordant with the results of PCR-RFLP and direct sequencing.
The HRM method developed here is an extremely sensitive, accurate and reliable technique and allows high-throughput, fast pre-screening to select for sequencing only those specimens that most likely contain mutant JAK2 V617F allele(s).

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    • "The melting curve analysis was carried out by the LightScanner software package with CALL-IT® software (Idaho Technology Inc., Salt Lake City, Utah, USA). Melting profiles were calibrated by internal oligonucleotide controls, and then normalized, grouped and displayed as fluorescence-versus-temperature plots or subtractive difference plots (−df/dt vs T) [5]. Heterozygous amplicons yielded recognizably different melting curves in comparison to homozygous samples which made it easy to identify the heterozygotes. "
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    ABSTRACT: Objective: Recurrent L265P mutation of myeloid differentiation primary response gene 88 (MYD88) has been identified in a proportion of diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia. The present study aimed to establish a rapid, sensitive, and reliable method using high-resolution melting analysis (HRMA) to detect MYD88 L265P mutation in DLBCL. Designs and methods: The sensitivity of HRMA in the detection of MYD88 L265P mutation was evaluated. MYD88 L265P mutation was further screened in 120 patients with DLBCL. The results of HRMA were validated by direct DNA sequencing. Results: For the detection of MYD88 L265P mutation, the reproducible maximal sensitivity of HRMA was 5% higher than that obtained by direct DNA sequencing (25%). Heterozygous MYD88 L265P mutations were identified in 11 (9.2%) DLBCL cases, all of which were diagnosed as non-germinal-center B cell (non-GCB) DLBCL. Conclusions: The HRMA assay is a rapid, sensitive, reliable, and high-throughput method to screen MYD88 L265P mutation and could be used in clinical diagnostic laboratories.
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    • "are listed in Table S1. genomic DNA samples were amplified in a final volume of 25 μl containing 1× PCR buffer (Invitrogen, Merelbeke, Belgium), 0.2 mmol/L of each dNTP, 2.5 mmol/L of MgCl2, 0.4 μmol/L of both forward and reverse primers, 0.8 μmol/L of oligonucleotide calibrators [13], 1× LCgreen Plus (Idaho Technology Inc. Salt Lake City, Utah), and 1 U Taq polymerase (MBI Fermentas, Canada). All PCR amplicons were generated on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA) using the PCR program as follows: an initial denaturation step was started at 95°C for 5 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at melting temperature for 30 seconds, and an extension at 72°C for 30 seconds. "
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    • "The size of PCR product was 67-bp. PCR was performed in 25-µL volume in the presence of 1× PCR buffer (Invitrogen, Merelbeke, Belgium), 0.2 mmol/L of each dNTP, 2.5 mmol/L of MgCl2, 0.4 µmol/L of both forward and reverse primers, 0.8 µmol/L of oligonucleotide calibrators [7], 1× LCgreen Plus (Idaho Technology Inc. Salt Lake City, Utah), 1 U Taq polymerase (MBI Fermentas, Canada), and 50 ng genomic DNA. PCR reactions were carried out on a 7300 Thermo cycler (Applied Biosystems, Foster City, CA, USA). "
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