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Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry

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Abstract

Quantification of genomic DNA that is traceable to the SI was performed successfully by measuring the individual nucleotides. Specifically, ultrasound was used to shear lambda genomic DNA into fragments of less than 200 base pairs, followed by deoxyribonuclease Ι and phosphodiesterase Ι digestion and liquid chromatography–isotope dilution mass spectrometry (LC-IDMS) quantification to estimate the mass fraction of the lambda DNA, based on the constituent deoxynucleotide monophosphates (dNMPs) within the molecule. Digital PCR (dPCR) was employed to quantify the same lambda DNA solution to provide independent data for comparing the performance of two quantitative methods. On the basis of the LC-IDMS measurement after ultrasonic treatment of the sample, the concentration of lambda DNA was 273.1 ± 9.8 μg/g (expanded uncertainty at the 95% confidence interval). This shows good agreement with the data from dPCR. Additionally, the result calculated on the basis of the sum of the concentrations of the four dNMPs is the same as that calculated on the basis of the sequence, which indicates that knowledge of the DNA sequence and length is unnecessary to measure the total DNA concentration when applying ultrasonic treatment–LC-IDMS.

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Acknowledgements

This work was supported by the Ministry of Science and Technology of China, as a part of a key project (no. 2008BAK41B01) in the National Science & Technology Pillar Program and a grant from the National major special project for the development of transgenic organisms (2008ZX08012-003). We thank Dai Xinhua and Daniel Burke for their valuable comments.

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  1. Peng Li

    Present address: Millipore China Limited Beijing Office, 18/F, Building A, Phoenix Place, No. A5 Shuguangxili, Chaoyang District, Beijing, 100028, China

Authors and Affiliations

  1. National Institute of Metrology, Beijing, 100013, China

    Lianhua Dong, Chao Zang, Jing Wang, Liang Li, Yunhua Gao, Liqing Wu & Peng Li

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  1. Lianhua Dong
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  2. Chao Zang
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  3. Jing Wang
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  4. Liang Li
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  5. Yunhua Gao
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  7. Peng Li
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Correspondence to Lianhua Dong.

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Dong, L., Zang, C., Wang, J. et al. Lambda genomic DNA quantification using ultrasonic treatment followed by liquid chromatography–isotope dilution mass spectrometry. Anal Bioanal Chem 402, 2079–2088 (2012). https://doi.org/10.1007/s00216-011-5644-5

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