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| Accurate Plasmid DNA Copy Concentration Quantification by Digital PCR |
| DONG Lian-hua,SUI Zhi-wei,WANG Jing,FU Bo-qiang |
| National Institute of Metrology, Beijing 100029, China |
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Abstract NIM established two digital polymerase chain reaction (dPCR) assays for quantifying the plasmid DNA sample, including designing two pairs of specific primer and probe, optimizing PCR amplifying conditions and excluding the DNA contamination in the PCR mastermix. The measurement results by simplex and duplex dPCR are compared. The DNA sample is successfully quantified by the established dPCR method and the measurement uncertainty is eventually evaluated. The measurement result with its expanded uncertainty (k=2) is (8.06±0.55)×103copies/mg, it agree well with the reference value within the standard uncertainty and very close to the reference value.
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Received: 09 November 2015
Published: 28 February 2017
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Corresponding Authors:
lianhua dong
E-mail: lianhuadong@126.com
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［1］White R A, Blainey P C, Fan H C, et al. Digital PCR provides sensitive and absolute calibration for high throughput sequencing ［J］. BMC Genomics,2009,10(1):116-121.
［2］Sanders R, Huggett J F, Bushell C A, et al. Evaluation of digital PCR for absolute DNA quantification［J］.Analytical Chemistry,2011, 83(17): 6474-6484.
［3］Dingle T C, Sedlak R H, Cook L, et al. Tolerance of droplet-digital PCR versus real-time quantitative PCR to inhibitory substances ［J］. Clinical Chemistry,2013, 59（11）: 1670-1672.
［4］Whale A S, Huggett J F, Cowen S, et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation ［J］.Nucleic Acids Research, 2012, 40（11）: e82.
［5］Corbisier P, Bhat S, Partis L, et al. Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction ［J］. Analytcial and Bioanalytical Chemistry,2010, 396(6): 2143-2150.
［6］Haynes R J, Kline M C, Toman B, et al. Standard reference material 2366 for measurement of human cytomegalovirus DNA ［J］.Journal of Molecular Diagnostic,2013, 15(2): 177-185.
［7］Dong L, Meng Y, Wang J, et al. Evaluation of droplet digital PCR on characterization of plasmid reference material for quantifying ammonia oxidizer and denitrifier ［J］.Analytcial and Bioanalytical Chemistry,2014, 406(20):1701-1712.
［8］Burke D G, Dong L, Bhat S, et al. Digital polymerase chain reaction measured pUC19 marker as calibrant for HPLC measurement of DNA quantity ［J］.Analytical Chemistry,2013, 85（3）:1657-1664.
［9］Tilburg J J, Nabuurs-Franssen M H, Hannen E J, et al.Contamination of commercial PCR master mix with DNA from Coxiellaburnetii ［J］.Journal of Clinical Microbiology,2010, 48（12）: 4634-4635.
［10］Pinheiro L B, Coleman V A, Hindson C M, et al. Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification ［J］.Analytical Chemistry, 2012,84（2）:1003-1011.
［11］Park S Y. CCQM-P154: Absolute Quantification of DNA［R］. CCQM study report,2016.
［12］Dong L H, Yoo H, Wang J, et al. Accurate quantification of supercoiled DNA by digital PCR［J］. Scientific Reports,2016,6:24230. |
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2017, Vol. 38 
