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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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Related in: MedlinePlus

Expression and activity of the high-purity φ29 DNAP. (A) Denaturing acrylamide gel following expression and purification of affinity-tagged φ29 DNAP. Lanes show content of crude lysate, column flow-throughs (FT) and eluates. The arrow indicates the expression target. Bands in product below 63 kDa marker in the final product (second Ni affinity elution) are most likely C-terminal deletions of the designed product: no effort was made to remove these. (B) Agarose gel showing MDA products from μl-scale reactions using 10 000 GE E. coli genomic DNA as template. The variously sourced enzymes all produced high molecular weight DNA products.
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Figure 1: Expression and activity of the high-purity φ29 DNAP. (A) Denaturing acrylamide gel following expression and purification of affinity-tagged φ29 DNAP. Lanes show content of crude lysate, column flow-throughs (FT) and eluates. The arrow indicates the expression target. Bands in product below 63 kDa marker in the final product (second Ni affinity elution) are most likely C-terminal deletions of the designed product: no effort was made to remove these. (B) Agarose gel showing MDA products from μl-scale reactions using 10 000 GE E. coli genomic DNA as template. The variously sourced enzymes all produced high molecular weight DNA products.

Mentions: We designed a synthetic gene encoding wild-type ϕ29 DNAP with dual, cleavable, N-terminal affinity tags (hexahistidine and glutathione S-transferase). The expression plasmid was transformed into E. coli, which was grown on the liter scale in DNAse-treated media and induced to over-produce the target enzyme by standard methods. The cells were lysed by sonication and lysozyme treatment and the lysate was spiked with DNAse I. The protein was purified by serial affinity chromatography steps on metal-affinity and glutathione resin. After an initial metal-affinity step with limiting resin, the preparation was treated with DNAse I and RNAse A before subsequent affinity purifications on the glutathione resin and fresh metal-affinity resin to remove the DNAse and RNAse endonuclease activities. Figure 1A shows a denaturing and reducing acrylamide gel following the steps of the purification. The major purification product occurs just below the 98 kDa marker, consistent with the expected 93 kDa molecular weight of the target construct. The bands in the purified product <63 kDa are most likely C-terminal deletions of the designed product: no effort was made to remove these. We note that affixing one tag to each end of the protein would enable selection of the full-length product using the same purification procedure. The purification netted 3.5 mg of the tagged product based on the absorbance of the sample at 280 nm.Figure 1.


Digital MDA for enumeration of total nucleic acid contamination.

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

Expression and activity of the high-purity φ29 DNAP. (A) Denaturing acrylamide gel following expression and purification of affinity-tagged φ29 DNAP. Lanes show content of crude lysate, column flow-throughs (FT) and eluates. The arrow indicates the expression target. Bands in product below 63 kDa marker in the final product (second Ni affinity elution) are most likely C-terminal deletions of the designed product: no effort was made to remove these. (B) Agarose gel showing MDA products from μl-scale reactions using 10 000 GE E. coli genomic DNA as template. The variously sourced enzymes all produced high molecular weight DNA products.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Expression and activity of the high-purity φ29 DNAP. (A) Denaturing acrylamide gel following expression and purification of affinity-tagged φ29 DNAP. Lanes show content of crude lysate, column flow-throughs (FT) and eluates. The arrow indicates the expression target. Bands in product below 63 kDa marker in the final product (second Ni affinity elution) are most likely C-terminal deletions of the designed product: no effort was made to remove these. (B) Agarose gel showing MDA products from μl-scale reactions using 10 000 GE E. coli genomic DNA as template. The variously sourced enzymes all produced high molecular weight DNA products.
Mentions: We designed a synthetic gene encoding wild-type ϕ29 DNAP with dual, cleavable, N-terminal affinity tags (hexahistidine and glutathione S-transferase). The expression plasmid was transformed into E. coli, which was grown on the liter scale in DNAse-treated media and induced to over-produce the target enzyme by standard methods. The cells were lysed by sonication and lysozyme treatment and the lysate was spiked with DNAse I. The protein was purified by serial affinity chromatography steps on metal-affinity and glutathione resin. After an initial metal-affinity step with limiting resin, the preparation was treated with DNAse I and RNAse A before subsequent affinity purifications on the glutathione resin and fresh metal-affinity resin to remove the DNAse and RNAse endonuclease activities. Figure 1A shows a denaturing and reducing acrylamide gel following the steps of the purification. The major purification product occurs just below the 98 kDa marker, consistent with the expected 93 kDa molecular weight of the target construct. The bands in the purified product <63 kDa are most likely C-terminal deletions of the designed product: no effort was made to remove these. We note that affixing one tag to each end of the protein would enable selection of the full-length product using the same purification procedure. The purification netted 3.5 mg of the tagged product based on the absorbance of the sample at 280 nm.Figure 1.

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