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Quantitative and sensitive detection of rare mutations using droplet-based microfluidics.
MedLine Citation:
PMID:  21594292     Owner:  NLM     Status:  Publisher    
Abstract/OtherAbstract:
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200 000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.
Authors:
Deniz Pekin; Yousr Skhiri; Jean-Christophe Baret; Delphine Le Corre; Linas Mazutis; Chaouki Ben Salem; Florian Millot; Abdeslam El Harrak; J Brian Hutchison; Jonathan W Larson; Darren R Link; Pierre Laurent-Puig; Andrew D Griffiths; Valérie Taly
Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-5-19
Journal Detail:
Title:  Lab on a chip     Volume:  -     ISSN:  1473-0189     ISO Abbreviation:  -     Publication Date:  2011 May 
Date Detail:
Created Date:  2011-5-19     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101128948     Medline TA:  Lab Chip     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
Affiliation:
Institut de Science et d'Ingénierie Supramoléculaires (ISIS), Université de Strasbourg, CNRS UMR 7006, 8 allée Gaspard Monge, BP 70028, F-67083, Strasbourg Cedex, France. griffiths@unistra.fr vtaly@unistra.fr.
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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