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Updated: Mar 10, 2026
Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids
Published on: June 8, 2019
Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and
Joonhong Park1, Minsik Song2, Woori Jang1
1Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea; Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
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.
