Rapid detection of JAK2 V617F mutation using high-resolution melting analysis with LightScanner platform.
ABSTRACT 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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ABSTRACT: A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.BioMed research international. 01/2014; 2014:458457.
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ABSTRACT: Hotspot mutations of serine/arginine-rich splicing factor 2 (SRSF2) gene have been identified in a proportion of hematologic malignancies including myelodysplastic syndrome (MDS). The aim of the present study was to develop a new approach to screen SRSF2 mutation and analyze the clinical relevance of SRSF2 mutations in Chinese MDS. A protocol based on high-resolution melting analysis (HRMA) was established to screen SRSF2-P95 mutation in 108 MDS patients and was compared with Sanger sequencing. The clinical relevance of SRSF2 mutations was further evaluated. HRMA identified five (4.6%) cases with SRSF2 mutation, completely validated by Sanger sequencing without false positive or negative results. The sensitivities of HRMA and Sanger sequencing were 10% and 25% for the detection of SRSF2-P95H mutation, respectively, against the background of wild-type DNA. Patients with SRSF2 mutation had shorter overall survival time than those with wild-type SRSF2 in both the whole cohort of cases and those with normal karyotype (P = 0.069 and 0.023, respectively). Multivariate analysis confirmed SRSF2 mutation as an independent risk factor in both patient populations. We established a fast, high-throughput, and inexpensive HRMA-based method to screen SRSF2 mutation, which could be used in clinical diagnostic laboratories. SRSF2 mutations were significantly associated with mortality rate in the MDS affected Chinese.PLoS ONE 12/2014; 9(12):e115693. · 3.53 Impact Factor
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ABSTRACT: The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the insulin-like growth factor I (IGF-I) gene and analyze the potential association between IGF-I gene polymorphisms and growth traits in a crossing sinipercid population. Two SNPs (g.A321G, g.A537G) within five exons of the IGF-I gene were tested for association with four growth traits in 250 individuals using the high-resolution melting method. The association analysis of SNPs of IGF-I gene with the four growth traits was carried out using General Linear Model estimation. Results indicated that SNP1 in exon3 of the IGF-I gene was significantly associated with body weight (P < 0.05), body length (P < 0.05) and body width (P < 0.05), and SNP2 in exon5 of the IGF-I gene was significantly associated with body weight (P < 0.05), body depth (P < 0.05) and body width (P < 0.05) in a sinipercid population. The individuals of genotype AA or AG in loci SNP1 and SNP2 grew faster than those of genotype GG. Diplotypes did not show significant effects on growth traits. These findings implied that the two SNPs of the IGF-I gene affecting growth traits could be potential quantitative trait nucleotides and would be useful genetic markers in the selection of some growth traits for developing improved culture lines of sinipercid species in China.Fisheries Science 79(3). · 0.86 Impact Factor