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Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number

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Abstract

Digital polymerase chain reaction (PCR) is a promising technique for estimating target DNA copy number. PCR solution is distributed throughout numerous partitions, and following amplification, target DNA copy number is estimated based on the proportion of partitions containing amplified DNA. Here, we identify approaches for obtaining reliable digital PCR data. Single molecule amplification efficiency was significantly improved following fragmentation of total DNA and bias in copy number estimates reduced by analysis of short intact target DNA fragments. Random and independent distribution of target DNA molecules throughout partitions, which is critical to accurate digital PCR measurement, was demonstrated by spatial distribution analysis. The estimated relative uncertainty for target DNA concentration was under 6% when analyzing five digital panels comprising 765 partitions each, provided the panels contained an average of 212 to 3,365 template molecules. Partition volume was a major component of this uncertainty estimate. These findings can be applied to other digital PCR studies to improve confidence in such measurements.

Digital PCR amplification plot (left) and panel read out (right) of HindIII-digested pIRMM69. pIRMM69 contains one HindIII restriction enzyme site outside the hmg and transgene fragments used as targets in PCR. Red boxes with white shade denote positive hits containing one or more target DNA molecules, and white boxes with grey shade refer to no target being amplified.

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Abbreviations

bp:

Base pair

Ct:

Cycle threshold

DNA:

Deoxyribonucleic acid

ERM:

European reference material

GM:

Genetically modified

hmg:

High mobility group protein A

NTC:

No template control

PCR:

Polymerase chain reaction

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Correspondence to Kerry R. Emslie.

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Bhat, S., Herrmann, J., Armishaw, P. et al. Single molecule detection in nanofluidic digital array enables accurate measurement of DNA copy number. Anal Bioanal Chem 394, 457–467 (2009). https://doi.org/10.1007/s00216-009-2729-5

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  • DOI: https://doi.org/10.1007/s00216-009-2729-5

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