Eprobe-mediated screening system for somatic mutations in the KRAS locus

Eprobe-mediated screening system for somatic mutations in the KRAS locus

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventi...

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Main Authors: ATSUMI, JUN, HANAMI, TAKESHI, ENOKIDA, YASUAKI, OGAWA, HIROOMI, DELOBEL, DIANE, MITANI, YASUMASA, KIMURA, YASUMASA, SOMA, TAKAHIRO, TAGAMI, MICHIHIRA, TAKASE, YOSHIAKI, ICHIHARA, TATSUO, TAKEYOSHI, IZUMI, USUI, KENGO, HAYASHIZAKI, YOSHIHIDE, SHIMIZU, KIMIHIRO
Format: Online
Language:English
Published: D.A. Spandidos 2015
Online Access:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431451/
id pubmed-4431451
recordtype oai_dc
spelling pubmed-44314512015-05-22 Eprobe-mediated screening system for somatic mutations in the KRAS locus ATSUMI, JUN HANAMI, TAKESHI ENOKIDA, YASUAKI OGAWA, HIROOMI DELOBEL, DIANE MITANI, YASUMASA KIMURA, YASUMASA SOMA, TAKAHIRO TAGAMI, MICHIHIRA TAKASE, YOSHIAKI ICHIHARA, TATSUO TAKEYOSHI, IZUMI USUI, KENGO HAYASHIZAKI, YOSHIHIDE SHIMIZU, KIMIHIRO Articles Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence ‘Eprobe’ capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05–0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications. D.A. Spandidos 2015-06 2015-03-30 /pmc/articles/PMC4431451/ /pubmed/25823645 http://dx.doi.org/10.3892/or.2015.3883 Text en Copyright © 2015, Spandidos Publications http://creativecommons.org/licenses/by/3.0 This is an open-access article licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported License. The article may be redistributed, reproduced, and reused for non-commercial purposes, provided the original source is properly cited.
repository_type Open Access Journal
institution_category Foreign Institution
institution US National Center for Biotechnology Information
building NCBI PubMed
collection Online Access
language English
format Online
author ATSUMI, JUN
HANAMI, TAKESHI
ENOKIDA, YASUAKI
OGAWA, HIROOMI
DELOBEL, DIANE
MITANI, YASUMASA
KIMURA, YASUMASA
SOMA, TAKAHIRO
TAGAMI, MICHIHIRA
TAKASE, YOSHIAKI
ICHIHARA, TATSUO
TAKEYOSHI, IZUMI
USUI, KENGO
HAYASHIZAKI, YOSHIHIDE
SHIMIZU, KIMIHIRO
spellingShingle ATSUMI, JUN
HANAMI, TAKESHI
ENOKIDA, YASUAKI
OGAWA, HIROOMI
DELOBEL, DIANE
MITANI, YASUMASA
KIMURA, YASUMASA
SOMA, TAKAHIRO
TAGAMI, MICHIHIRA
TAKASE, YOSHIAKI
ICHIHARA, TATSUO
TAKEYOSHI, IZUMI
USUI, KENGO
HAYASHIZAKI, YOSHIHIDE
SHIMIZU, KIMIHIRO
Eprobe-mediated screening system for somatic mutations in the KRAS locus
author_facet ATSUMI, JUN
HANAMI, TAKESHI
ENOKIDA, YASUAKI
OGAWA, HIROOMI
DELOBEL, DIANE
MITANI, YASUMASA
KIMURA, YASUMASA
SOMA, TAKAHIRO
TAGAMI, MICHIHIRA
TAKASE, YOSHIAKI
ICHIHARA, TATSUO
TAKEYOSHI, IZUMI
USUI, KENGO
HAYASHIZAKI, YOSHIHIDE
SHIMIZU, KIMIHIRO
author_sort ATSUMI, JUN
title Eprobe-mediated screening system for somatic mutations in the KRAS locus
title_short Eprobe-mediated screening system for somatic mutations in the KRAS locus
title_full Eprobe-mediated screening system for somatic mutations in the KRAS locus
title_fullStr Eprobe-mediated screening system for somatic mutations in the KRAS locus
title_full_unstemmed Eprobe-mediated screening system for somatic mutations in the KRAS locus
title_sort eprobe-mediated screening system for somatic mutations in the kras locus
description Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence ‘Eprobe’ capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05–0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.
publisher D.A. Spandidos
publishDate 2015
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431451/
_version_ 1613223459834298368

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