Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.

Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and fac...

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Autores principales:	Rajesh Patel, Alison Tsan, Rachel Tam, Rupal Desai, Jill Spoerke, Nancy Schoenbrunner, Thomas W Myers, Keith Bauer, Edward Smith, Rajiv Raja
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/c769aec52df6435e801952ca04055afa
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spelling	oai:doaj.org-article:c769aec52df6435e801952ca04055afa2021-11-18T08:04:52ZMutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.1932-620310.1371/journal.pone.0051153https://doaj.org/article/c769aec52df6435e801952ca04055afa2012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23284662/?tool=EBIhttps://doaj.org/toc/1932-6203Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific PCR (AS-PCR) assays. We analyzed a set of 71 mutations across six genes of therapeutic interest. The six-gene mutation panel was designed to detect the most common mutations in the EGFR, KRAS, PIK3CA, NRAS, BRAF, and AKT1 oncogenes. The DNA was preamplified using custom-designed primer sets before the TaqMan/AS-PCR assays were carried out using the Biomark microfluidics system (Fluidigm; South San Francisco, CA). A cross-reactivity analysis enabled the generation of a robust automated mutation-calling algorithm which was then validated in a series of 51 cell lines and 33 FFPE clinical samples. All detected mutations were confirmed by other means. Sample input titrations confirmed the assay sensitivity with as little as 2 ng gDNA, and demonstrated excellent inter- and intra-chip reproducibility. Parallel analysis of 92 clinical trial samples was carried out using 2-100 ng genomic DNA (gDNA), allowing the simultaneous detection of multiple mutations. DNA prepared from both fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples were used, and the analysis was routinely completed in 2-3 days: traditional assays require 0.5-1 µg high-quality DNA, and take significantly longer to analyze. This assay can detect a wide range of mutations in therapeutically relevant genes from very small amounts of sample DNA. As such, the mutation assay developed is a valuable tool for high-throughput biomarker discovery and validation in personalized medicine and cancer drug development.Rajesh PatelAlison TsanRachel TamRupal DesaiJill SpoerkeNancy SchoenbrunnerThomas W MyersKeith BauerEdward SmithRajiv RajaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 12, p e51153 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rajesh Patel Alison Tsan Rachel Tam Rupal Desai Jill Spoerke Nancy Schoenbrunner Thomas W Myers Keith Bauer Edward Smith Rajiv Raja Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
description	Targeted anticancer therapies rely on the identification of patient subgroups most likely to respond to treatment. Predictive biomarkers play a key role in patient selection, while diagnostic and prognostic biomarkers expand our understanding of tumor biology, suggest treatment combinations, and facilitate discovery of novel drug targets. We have developed a high-throughput microfluidics method for mutation detection (MUT-MAP, mutation multi-analyte panel) based on TaqMan or allele-specific PCR (AS-PCR) assays. We analyzed a set of 71 mutations across six genes of therapeutic interest. The six-gene mutation panel was designed to detect the most common mutations in the EGFR, KRAS, PIK3CA, NRAS, BRAF, and AKT1 oncogenes. The DNA was preamplified using custom-designed primer sets before the TaqMan/AS-PCR assays were carried out using the Biomark microfluidics system (Fluidigm; South San Francisco, CA). A cross-reactivity analysis enabled the generation of a robust automated mutation-calling algorithm which was then validated in a series of 51 cell lines and 33 FFPE clinical samples. All detected mutations were confirmed by other means. Sample input titrations confirmed the assay sensitivity with as little as 2 ng gDNA, and demonstrated excellent inter- and intra-chip reproducibility. Parallel analysis of 92 clinical trial samples was carried out using 2-100 ng genomic DNA (gDNA), allowing the simultaneous detection of multiple mutations. DNA prepared from both fresh frozen and formalin-fixed, paraffin-embedded (FFPE) samples were used, and the analysis was routinely completed in 2-3 days: traditional assays require 0.5-1 µg high-quality DNA, and take significantly longer to analyze. This assay can detect a wide range of mutations in therapeutically relevant genes from very small amounts of sample DNA. As such, the mutation assay developed is a valuable tool for high-throughput biomarker discovery and validation in personalized medicine and cancer drug development.
format	article
author	Rajesh Patel Alison Tsan Rachel Tam Rupal Desai Jill Spoerke Nancy Schoenbrunner Thomas W Myers Keith Bauer Edward Smith Rajiv Raja
author_facet	Rajesh Patel Alison Tsan Rachel Tam Rupal Desai Jill Spoerke Nancy Schoenbrunner Thomas W Myers Keith Bauer Edward Smith Rajiv Raja
author_sort	Rajesh Patel
title	Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
title_short	Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
title_full	Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
title_fullStr	Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
title_full_unstemmed	Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.
title_sort	mutation scanning using mut-map, a high-throughput, microfluidic chip-based, multi-analyte panel.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/c769aec52df6435e801952ca04055afa
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Mutation scanning using MUT-MAP, a high-throughput, microfluidic chip-based, multi-analyte panel.

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