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A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

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 en...

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Autores principales: Wu, Zhiyuan, Zhang, Yunqing, Zhang, Xinju, Xu, Xiao, Kang, Zhihua, Li, Shibao, Zhang, Chen, Su, Bing, Guan, Ming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960525/
https://www.ncbi.nlm.nih.gov/pubmed/24729973
http://dx.doi.org/10.1155/2014/458457
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author Wu, Zhiyuan
Zhang, Yunqing
Zhang, Xinju
Xu, Xiao
Kang, Zhihua
Li, Shibao
Zhang, Chen
Su, Bing
Guan, Ming
author_facet Wu, Zhiyuan
Zhang, Yunqing
Zhang, Xinju
Xu, Xiao
Kang, Zhihua
Li, Shibao
Zhang, Chen
Su, Bing
Guan, Ming
author_sort Wu, Zhiyuan
collection PubMed
description 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.
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spelling pubmed-39605252014-04-13 A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms Wu, Zhiyuan Zhang, Yunqing Zhang, Xinju Xu, Xiao Kang, Zhihua Li, Shibao Zhang, Chen Su, Bing Guan, Ming Biomed Res Int Research Article 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. Hindawi Publishing Corporation 2014 2014-03-05 /pmc/articles/PMC3960525/ /pubmed/24729973 http://dx.doi.org/10.1155/2014/458457 Text en Copyright © 2014 Zhiyuan Wu et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Wu, Zhiyuan
Zhang, Yunqing
Zhang, Xinju
Xu, Xiao
Kang, Zhihua
Li, Shibao
Zhang, Chen
Su, Bing
Guan, Ming
A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title_full A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title_fullStr A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title_full_unstemmed A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title_short A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms
title_sort multiplex snapback primer system for the enrichment and detection of jak2 v617f and mpl w515l/k mutations in philadelphia-negative myeloproliferative neoplasms
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3960525/
https://www.ncbi.nlm.nih.gov/pubmed/24729973
http://dx.doi.org/10.1155/2014/458457
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