Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification
| Autor: | Leonardo B. Pinheiro, Christopher Hindson, Benjamin J. Hindson, Kerry R. Emslie, Victoria A. Coleman, Somanath Bhat, Jan Herrmann |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
DNA Copy Number Variations
01 natural sciences Polymerase Chain Reaction Article Analytical Chemistry law.invention 03 medical and health sciences chemistry.chemical_compound law Digital polymerase chain reaction Polymerase chain reaction Digital droplet pcr 030304 developmental biology 0303 health sciences Genome 010401 analytical chemistry Dna concentration High-Throughput Nucleotide Sequencing DNA Molecular biology Bacteriophage lambda 0104 chemical sciences 3. Good health chemistry Microfluidic chamber Nucleic acid Biological system |
| Zdroj: | Analytical Chemistry |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies. |
| Databáze: | OpenAIRE |
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