| COLD PCR HRM: a highly sensitive detection method for IDH1 mutations. | |
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MedLine Citation:
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PMID: 20886613 Owner: NLM Status: In-Process |
Abstract/OtherAbstract:
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The p.Arg132His mutation of isocitrate dehydrogenase 1 (IDH1(R132H) ) is a frequent alteration and a major prognostic marker in gliomas. However, direct sequencing of highly contaminated tumor samples may fail to detect this mutation. Our objective was to evaluated the sensitivity of a newly described amplification method, coamplification at lower temperature-PCR (COLD PCR), combined with high-resolution melting (HRM) for the detection of the IDH1(R132H) mutation. To this end, we used serial dilutions of mutant DNA with wild-type DNA. PCR-HRM assay detects IDH1(R132H) at an abundance of 25%, similar to the detection limit of direct Sanger sequencing. Introducing a run of COLD PCR allows the detection of 2% mutant DNA. Using two consecutive runs of COLD PCR, we detected 0.25% mutant DNA in a background of wild-type DNA, that mimics a tumor sample highly contaminated by normal DNA. We then analyzed 10 biopsies of tumor edges, considered free of tumor cells by histological analysis, and showed that immunohistochemistry of IDH1(R132H) was positive in three cases (30%), whereas double COLD PCR HRM was positive in the 10 cases studied (100%). In summary, COLD PCR HRM analysis is 100-fold more sensitive than Sanger sequencing, rendering this rapid and powerful strategy particularly useful for samples highly contaminated with normal tissue. |
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Authors:
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Blandine Boisselier; Yannick Marie; Marianne Labussière; Pietro Ciccarino; Virginie Desestret; XiaoWei Wang; Laurent Capelle; Jean-Yves Delattre; Marc Sanson |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2010-11-09 |
Journal Detail:
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Title: Human mutation Volume: 31 ISSN: 1098-1004 ISO Abbreviation: Hum. Mutat. Publication Date: 2010 Dec |
Date Detail:
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Created Date: 2010-11-24 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9215429 Medline TA: Hum Mutat Country: United States |
Other Details:
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Languages: eng Pagination: 1360-5 Citation Subset: IM |
Copyright Information:
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© 2010 Wiley-Liss, Inc. |
Affiliation:
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Université Pierre et Marie Curie-Paris 6, Centre de Recherche de l'Institut du Cerveau et de la Moëlle épinière (CRICM) UMR-S975, Paris, France. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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