title: Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.
abstract: 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.
authors.affiliation: Department 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.
authors.forename: Joonhong
authors.initials: J
authors.lastname: Park
authors.affiliation: SeaSun Biomaterials, Daejeon, Republic of Korea.
authors.forename: Minsik
authors.initials: M
authors.lastname: Song
authors.affiliation: Department 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.
authors.forename: Woori
authors.initials: W
authors.lastname: Jang
authors.affiliation: Department 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.
authors.forename: Hyojin
authors.initials: H
authors.lastname: Chae
authors.affiliation: Catholic Genetic Laboratory Center, Seoul St. Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
authors.forename: Gun Dong
authors.initials: GD
authors.lastname: Lee
authors.affiliation: SeaSun Biomaterials, Daejeon, Republic of Korea.
authors.forename: KyungTak
authors.initials: K
authors.lastname: Kim
authors.affiliation: SeaSun Biomaterials, Daejeon, Republic of Korea.
authors.forename: Heekyung
authors.initials: H
authors.lastname: Park
authors.affiliation: Department 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. Electronic address: microkim@catholic.ac.kr.
authors.forename: Myungshin
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department 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. Electronic address: yonggoo@catholic.ac.kr.
authors.forename: Yonggoo
authors.initials: Y
authors.lastname: Kim
mesh_terms: D000368:Aged; D037282:Calreticulin / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005453:Fluorescence; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D054437:Myelodysplastic-Myeloproliferative Diseases / Q000235:genetics; D020135:Peptide Nucleic Acids; D053628:Receptors, Thrombopoietin / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: C576483:CALR protein, human; D037282:Calreticulin; D020135:Peptide Nucleic Acids; D053628:Receptors, Thrombopoietin; C082218:MPL protein, human; D016044:Fusion Proteins, bcr-abl; C507924:JAK2 protein, human; D053614:Janus Kinase 2
keywords: CALR; FMCA; JAK2; MPL; Mutation screening; Peptide nucleic acid