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Digital MDA for enumeration of total nucleic acid contamination.

Blainey PC, Quake SR - Nucleic Acids Res. (2010)

Bottom Line: Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP).Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence.Contamination in commercially available preparations is also investigated.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Stanford, CA 94305, USA.

ABSTRACT
Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

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Digital MDA on Fluidigm 12.765 digital array reveals varying levels of contamination in φ29 DNAP from three commercial providers. φ29 DNAP prepared in-house shows little contamination. A single reaction mix including exceptionally clean lots of the GE MDA ‘reaction’ and ‘sample’ buffers were used in all cases. (A) Example image of dMDA assay endpoint (all four panels are from a single chip run). Readout is digital, indicating contaminants, but not primer-derived products, underlie background amplification. (B) Quantification of dMDA assay results, indicating contaminant levels in the various enzyme preparations. The boxplots show data quartile ranges, median (black line) and outlier values (crosses). Results from several enzyme lots are pooled for each manufacturer, with 9–13 total dMDA assays per enzyme source.
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Figure 3: Digital MDA on Fluidigm 12.765 digital array reveals varying levels of contamination in φ29 DNAP from three commercial providers. φ29 DNAP prepared in-house shows little contamination. A single reaction mix including exceptionally clean lots of the GE MDA ‘reaction’ and ‘sample’ buffers were used in all cases. (A) Example image of dMDA assay endpoint (all four panels are from a single chip run). Readout is digital, indicating contaminants, but not primer-derived products, underlie background amplification. (B) Quantification of dMDA assay results, indicating contaminant levels in the various enzyme preparations. The boxplots show data quartile ranges, median (black line) and outlier values (crosses). Results from several enzyme lots are pooled for each manufacturer, with 9–13 total dMDA assays per enzyme source.

Mentions: To directly compare contaminant levels in different preparations of ϕ29 DNAP, we ran NTC dMDA assays using enzyme from four sources and the same reaction buffer employed in Figure 2. The results were quantified by counting spots and are plotted in Figure 3B, which reveals high contamination levels in the enzyme samples obtained from all three manufacturers and very low levels in our high-purity sample. We also made a more stringent test for contamination in the high-purity ϕ29 DNAP sample by over-loading a digital MDA assay with a 10-fold higher concentration of the high-purity enzyme. In order to maintain parity in enzyme activity, the excess ϕ29 DNAP was heat-killed (65°C for 10 min). No positive spots were detected, indicating that the high-purity ϕ29 DNAP has fewer than one MDA-active contaminating fragment per 500 U. This also implies that nonzero contaminant counts observed in NTC assays with the high-purity ϕ29 DNAP preparation (visible in Figure 2, ‘high-purity’ NTC and Figure 3B, ‘high-purity’) most likely arise from a source other than the high-purity enzyme.Figure 3.


Digital MDA for enumeration of total nucleic acid contamination.

Blainey PC, Quake SR - Nucleic Acids Res. (2010)

Digital MDA on Fluidigm 12.765 digital array reveals varying levels of contamination in φ29 DNAP from three commercial providers. φ29 DNAP prepared in-house shows little contamination. A single reaction mix including exceptionally clean lots of the GE MDA ‘reaction’ and ‘sample’ buffers were used in all cases. (A) Example image of dMDA assay endpoint (all four panels are from a single chip run). Readout is digital, indicating contaminants, but not primer-derived products, underlie background amplification. (B) Quantification of dMDA assay results, indicating contaminant levels in the various enzyme preparations. The boxplots show data quartile ranges, median (black line) and outlier values (crosses). Results from several enzyme lots are pooled for each manufacturer, with 9–13 total dMDA assays per enzyme source.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045575&req=5

Figure 3: Digital MDA on Fluidigm 12.765 digital array reveals varying levels of contamination in φ29 DNAP from three commercial providers. φ29 DNAP prepared in-house shows little contamination. A single reaction mix including exceptionally clean lots of the GE MDA ‘reaction’ and ‘sample’ buffers were used in all cases. (A) Example image of dMDA assay endpoint (all four panels are from a single chip run). Readout is digital, indicating contaminants, but not primer-derived products, underlie background amplification. (B) Quantification of dMDA assay results, indicating contaminant levels in the various enzyme preparations. The boxplots show data quartile ranges, median (black line) and outlier values (crosses). Results from several enzyme lots are pooled for each manufacturer, with 9–13 total dMDA assays per enzyme source.
Mentions: To directly compare contaminant levels in different preparations of ϕ29 DNAP, we ran NTC dMDA assays using enzyme from four sources and the same reaction buffer employed in Figure 2. The results were quantified by counting spots and are plotted in Figure 3B, which reveals high contamination levels in the enzyme samples obtained from all three manufacturers and very low levels in our high-purity sample. We also made a more stringent test for contamination in the high-purity ϕ29 DNAP sample by over-loading a digital MDA assay with a 10-fold higher concentration of the high-purity enzyme. In order to maintain parity in enzyme activity, the excess ϕ29 DNAP was heat-killed (65°C for 10 min). No positive spots were detected, indicating that the high-purity ϕ29 DNAP has fewer than one MDA-active contaminating fragment per 500 U. This also implies that nonzero contaminant counts observed in NTC assays with the high-purity ϕ29 DNAP preparation (visible in Figure 2, ‘high-purity’ NTC and Figure 3B, ‘high-purity’) most likely arise from a source other than the high-purity enzyme.Figure 3.

Bottom Line: Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP).Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence.Contamination in commercially available preparations is also investigated.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Stanford University and Howard Hughes Medical Institute, 318 Campus Drive, Stanford, CA 94305, USA.

ABSTRACT
Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by φ29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom φ29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

Show MeSH
Related in: MedlinePlus