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Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.

Dong L, Meng Y, Wang J, Liu Y - Anal Bioanal Chem (2014)

Bottom Line: The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR.Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR.The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Metrology, Beijing, 100013, China, donglh@nim.ac.cn.

ABSTRACT
DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

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Evaluation of the droplet digital PCR method for quantification of linearized plasmid pNIM-003, digested by EcoR1 with AOB PCR assay
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Fig5: Evaluation of the droplet digital PCR method for quantification of linearized plasmid pNIM-003, digested by EcoR1 with AOB PCR assay

Mentions: The ddPCR response over concentrations ranging from approximately 2.3–2,177,456 copies 20 μL−1 of ddPCR is shown in Fig. 5. The average number of accepted droplet events for all 30 reactions was 13,865, with a standard deviation of 2,449 (Fig. 5a), indicating successful droplet generation for all reactions. Because of the dead volume of the ddPCR reader, it is unable to estimate the true number of DNA targets if the number of droplets is too small when the DNA concentration is very low. Although there were two reactions with fewer than 10,000 droplets (see the arrows in Fig. 5a), this was not a problem because the concentration of DNA targets was high enough to generate a sufficient number of positive droplets. One-dimensional scatter plots of fluorescent droplet amplitudes for selected wells are shown in Fig. 5b. With restriction digestion, the positive droplets separated clearly from the negative droplets. However, the smear between positive and negative droplets still existed for the NECs (Fig. S2), suggesting unsuccessful amplification in initial cycles for these droplets. This is one factor causing underestimation of the true number of molecules by non-digestion treatment. Theoretically, there should be no negative droplets when the DNA concentration is high enough to saturate the generated droplets. In practice, this is true for the digested treatment (dilution S1 in Fig. 5a) but there are still negative droplets in the undigested plasmid (dilution S1 in Fig. S2), indicating unsuccessful single-molecule amplification of the non-linearized plasmid. This is another factor causing underestimation of the copy numbers of undigested plasmid.Fig. 5


Evaluation of droplet digital PCR for characterizing plasmid reference material used for quantifying ammonia oxidizers and denitrifiers.

Dong L, Meng Y, Wang J, Liu Y - Anal Bioanal Chem (2014)

Evaluation of the droplet digital PCR method for quantification of linearized plasmid pNIM-003, digested by EcoR1 with AOB PCR assay
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3936116&req=5

Fig5: Evaluation of the droplet digital PCR method for quantification of linearized plasmid pNIM-003, digested by EcoR1 with AOB PCR assay
Mentions: The ddPCR response over concentrations ranging from approximately 2.3–2,177,456 copies 20 μL−1 of ddPCR is shown in Fig. 5. The average number of accepted droplet events for all 30 reactions was 13,865, with a standard deviation of 2,449 (Fig. 5a), indicating successful droplet generation for all reactions. Because of the dead volume of the ddPCR reader, it is unable to estimate the true number of DNA targets if the number of droplets is too small when the DNA concentration is very low. Although there were two reactions with fewer than 10,000 droplets (see the arrows in Fig. 5a), this was not a problem because the concentration of DNA targets was high enough to generate a sufficient number of positive droplets. One-dimensional scatter plots of fluorescent droplet amplitudes for selected wells are shown in Fig. 5b. With restriction digestion, the positive droplets separated clearly from the negative droplets. However, the smear between positive and negative droplets still existed for the NECs (Fig. S2), suggesting unsuccessful amplification in initial cycles for these droplets. This is one factor causing underestimation of the true number of molecules by non-digestion treatment. Theoretically, there should be no negative droplets when the DNA concentration is high enough to saturate the generated droplets. In practice, this is true for the digested treatment (dilution S1 in Fig. 5a) but there are still negative droplets in the undigested plasmid (dilution S1 in Fig. S2), indicating unsuccessful single-molecule amplification of the non-linearized plasmid. This is another factor causing underestimation of the copy numbers of undigested plasmid.Fig. 5

Bottom Line: The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR.Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR.The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements.

View Article: PubMed Central - PubMed

Affiliation: National Institute of Metrology, Beijing, 100013, China, donglh@nim.ac.cn.

ABSTRACT
DNA reference materials of certified value have a critical function in many analytical processes of DNA measurement. Quantification of amoA genes in ammonia oxidizing bacteria (AOB) and archaea (AOA), and of nirS and nosZ genes in the denitrifiers is very important for determining their distribution and abundance in the natural environment. A plasmid reference material containing nirS, nosZ, amoA-AOB, and amoA-AOA is developed to provide a DNA standard with copy number concentration for ensuring comparability and reliability of quantification of these genes. Droplet digital PCR (ddPCR) was evaluated for characterization of the plasmid reference material. The result revealed that restriction endonuclease digestion of plasmids can improve amplification efficiency and minimize the measurement bias of ddPCR. Compared with the conformation of the plasmid, the size of the DNA fragment containing the target sequence and the location of the restriction site relative to the target sequence are not significant factors affecting plasmid quantification by ddPCR. Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) was used to provide independent data for quantifying the plasmid reference material. The copy number concentration of the digested plasmid determined by ddPCR agreed well with that determined by LC-IDMS, improving both the accuracy and reliability of the plasmid reference material. The reference value, with its expanded uncertainty (k = 2), of the plasmid reference material was determined to be (5.19 ± 0.41) × 10(9) copies μL(-1) by averaging the results of two independent measurements. Consideration of the factors revealed in this study can improve the reliability and accuracy of ddPCR; thus, this method has the potential to accurately quantify DNA reference materials.

Show MeSH