Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR
| Autor: | Valérie Taly, Jeffrey L. Olson, Darren R. Link, Steve K. Kotsopoulos, Smiti Bhattacharya, Andrew D. Griffiths, Qun Zhong, Jonathan W. Larson |
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| Přispěvatelé: | RainDance Technologies, Inc. [Lexington, MA, USA], Institut de Science et d'ingénierie supramoléculaires (ISIS), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA), TALY, Valerie, Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Réseau nanophotonique et optique, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS) |
| Rok vydání: | 2011 |
| Předmět: |
[SDV]Life Sciences [q-bio]
Microfluidics Biomedical Engineering Gene Dosage Color Bioengineering Computational biology 01 natural sciences Biochemistry Polymerase Chain Reaction Polymorphism Single Nucleotide Cell Line 03 medical and health sciences chemistry.chemical_compound Genotype Humans Multiplex Digital polymerase chain reaction Taq Polymerase Polymerase 030304 developmental biology 0303 health sciences biology 010401 analytical chemistry Reproducibility of Results General Chemistry Molecular biology 0104 chemical sciences [SDV] Life Sciences [q-bio] Real-time polymerase chain reaction Spectrometry Fluorescence chemistry Nucleic acid biology.protein DNA |
| Zdroj: | Lab on a Chip Lab on a Chip, Royal Society of Chemistry, 2011, 11 (13), pp.2167. ⟨10.1039/c1lc20126c⟩ Lab on a Chip, 2011, 11 (13), pp.2167. ⟨10.1039/c1lc20126c⟩ |
| ISSN: | 1473-0189 1473-0197 |
| DOI: | 10.1039/c1lc20126c⟩ |
| Popis: | International audience; Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented. |
| Databáze: | OpenAIRE |
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