Digital assays for nucleic acid detection work by direct or indirect counting of analyte molecules and require single-molecule detection sensitivity. Digital assays requiring enzymatic amplification to generate higher signal levels for counting rely on the physical segregation of analyte molecules by limiting dilution such that individual analyte molecules can be separately amplified and counted (19
). This is the case for both dPCR (20
) and dMDA. We have developed a quantitative and sensitive method for assaying nucleic acid contamination based on carrying out a large number of single-molecule MDA reactions in a microfluidic device. Termed digital MDA in analog to digital PCR, the technique is useful to enumerate the number of high molecular weight MDA-active template molecules of unknown sequence in a sample.
Until development of the dMDA assay, we found the quantification of contaminating bacterial DNA in MDA reagents to be surprisingly difficult. In principle, real-time MDA could be used to quantify contaminates, although calibrating such an assay presents a problem when the template characteristics are unknown. We could achieve good sensitivity by running universal 16S QPCR on the products of NTC MDA reactions. However, because the degree of pre-amplification by MDA was unknown (both in the gross sense and in a locus-specific sense as a consequence of MDA amplification bias), the result was not quantitative and could not be effectively used to compare different reagent sets or the effects of different reagent treatments. On the other hand, if we strove for a fully quantitative readout by direct QPCR, we found no consistent signal above the baseline amplification of microbial DNA from the PCR reagents themselves. Microfluidic dPCR addresses the problem of interference by PCR reagent contamination by reducing the assay volume, but with an unacceptable tradeoff in detection sensitivity. For example, even under ideal conditions, 1000 GE of E. coli
DNA per milliliter in 10
kb fragments will give only two spots per panel on the 12.765 digital array (in PCR mode) and would be undetectable on the 48.770 digital array (in PCR mode).
Because the ssu rRNA gene makes up <1% of many bacterial genomes, there is a significant sensitivity gain to be had in using the MDA reaction rather than PCR for fragment detection, since any fragment, rather than just ssu rRNA gene-containing fragments, can be detected by MDA. For determination of MDA reagent contamination, there are the further advantages that no interference from additional reagents need be introduced and that the presence of MDA-inactive substrates is of no consequence since they cannot compete with amplification of desired templates, although MDA-inactive substrates would formally contribute to false-negative counts in quantification applications. The performance advantage of dMDA over dPCR is sizable for assays of highly fragmented or damaged DNA, as demonstrated in and expected in specific application areas such as forensics, studies of ancient DNA and astrobiology.
The high-purity ϕ29 DNAP reagent is useful not only to support the dMDA quantification method, but also for our original goal of carrying out single-cell MDA reactions free of contamination. indicates the expected number of contaminating DNA fragments in MDA reactions at three scales: 6, 60 and 6000
nl. The dMDA reactions presented in this paper are 6
nl in volume, while our microfluidic single-cell MDA reactions are typically 60
nl in volume, and 6
μl represents the smallest MDA reactions typically set up manually or with fluid-handling robots. Using the commercial enzyme preparations, all of the 6
μl MDA reactions will be contaminated, while more than half of the 60
nl MDA reactions could be contaminant-free, depending on the manufacturer chosen and the particular enzyme lot. We and others have made the disconcerting observation that contaminants in the commercial MDA reagents are not limited to the expression host (presumably an E. coli
B strain), but are rather drawn from an eclectic, albeit stereotyped group of bacterial species, making informatic subtraction of contaminating sequences an unattractive solution.
Contaminating DNA fragment number as function of enzyme batch and MDA reaction volume, based on median fragment values shown in B and the assumption that all reagents other than the enzyme are free of contaminates
With the high-purity enzyme, even microliter-scale MDA reactions free of contamination are possible. The commercial suppliers of MDA reagents typically specify 10
ng as the minimum quantity of template material. This limitation is not imposed by the sensitivity of MDA, but rather by the competing amplification of contaminants that reduce the amplification yield from the intended template material. The ability to amplify smaller amounts of template contaminant-free is a critical capability for single-cell genomics and other applications where a small amount of template material needs to be specifically amplified in a sequence-independent manner.
In the 12.765 digital array, with a clean ϕ29 DNAP preparation, the dMDA limit of detection is below 1 fragment per
μl and the limit of quantification (10 positives per d12 panel) is two fragments per microliter (~1
fg/ml for 2
kb fragments) or about one bacterial GE per microliter given uniformly-sized fragments. The fact that we see a somewhat larger number of spots than expected for uniform fragments indicates that smaller fragments exist in our E. coli
genomic sample, that dMDA can detect these smaller fragments with high efficiency, and that the practical quantification limit for highly fragmented bacterial DNA is much better than one GE per microliter. The LOD and LOQ values can be pushed even lower by using more than one panel per sample on the 12.765 chips or by increasing the assay volume in a different microfluidic device or emulsion-based platform. Assuring a low reagent background, including low contamination of the DNA polymerase, is key to realize the improved sensitivity in a larger assay volume. Conversely, shrinking the compartment volume is called for in dMDA quantification of nucleic acids at higher concentrations without dilution.
The LOQ <1
fg/ml demonstrated here for dMDA compares favorably with chemical, immunologic and PCR-based assays for organismal DNA. With respect to PCR detection of DNA fragments, the factor by which dMDA is expected to outperform dPCR depends on several features of the analyte, including PCR target locus density, the DNA fragment size and the template quality. For example, taking bacterial DNA in 4
kb fragments, with a PCR target locus density of 1 per Megabyte (typical of ssu rRNA genes), under idealized circumstances, there exist 250 analyte molecules for dMDA for each PCR-active analyte molecule, implying two to three orders of magnitude better sensitivity for the MDA-based assay. However, our real-world data show an even greater performance advantage for dMDA (). This is due to the fact that dMDA comes closer to its theoretical potential than does dPCR on the sheared E. coli
test sample. First, the shearing operation creates double-stranded breaks at random locations in the template DNA, reducing the fraction of fragments carrying the intact target locus for PCR. Second, it is likely that nicks are introduced by the harsh shearing condition in addition to double-stranded breaks. Since we carried out no steps to repair the template and diluted the template at low ionic strength, the presence of nicks is likely to reduce further the fraction of PCR-amplifiable fragments. The requirements for intact target loci and longer priming sites render PCR-based quantification more sensitive to template quality than MDA-based assays.
Digital MDA also provides new insights into the MDA process itself. For instance, we can test hypotheses about the source(s) of background amplification in microliter-scale MDA reactions. The digital nature of the dMDA results (i.e. bimodal distribution of spot intensities) and template concentration-dependence in observed in experiments with E. coli
genomic DNA fragments strongly implicate high molecular weight contaminants, but not primer-primer interactions, as the source of background amplification. The bimodal distribution of spot intensities cannot be explained by varying combinations of random primer sequences in different nanoliter MDA reactions. For perfectly random hexamers at 50
μM, the 6
nl wells in the 12.765 digital array each contain 150 billion primer molecules. Since there are 4096 6-mer sequence variants, each sequence is represented by nearly 50 million copies in each reaction well. According to random sampling statistics, the relative variation in concentration of sequence variants from well to well is <0.02%. Such small differences in representation are unlikely to explain the observed variation of SYBR green fluorescence intensity in the dMDA assay. The fact that the background level of SYBR green in negative spots does not increase over the course of the reaction is also inconsistent with the generation of high molecular weight product from primers alone in the GE and Qiagen reaction mixes. Thus, we are able to show that the background amplification indeed arises from high molecular weight contaminants and is not intrinsic to the MDA reagents.
This realization, in combination with the application of dMDA as a means of counting nucleic acid fragments in a sequence-independent manner, opens up new possibilities for the ultra-sensitive detection of DNA in various contexts. Often, in assays for the presence of microbes, ssu rRNA-encoding DNA serves as a proxy analyte for the organisms themselves. Digital MDA promises to improve the sensitivity of such assays by orders of magnitude in contexts where the loss of sequence-specificity can be tolerated or constitutes an advantage. The dMDA assay extends several features of the dPCR assay to MDA, including absolute quantification that requires no standard curve. Digital MDA demonstrates superior sensitivity to dPCR in this application because every MDA-active DNA fragment is detected, rather than some fraction containing a particular, intact, sequence locus (which may be a tiny fraction). Digital MDA of whole genomes or chromosomes from cells represents the ultimate goal of sample preparation for single-cell genomics. Capabilities for cell lysis and product recovery need to be integrated to access this application. Forthcoming innovations in hardware platforms intended for digital PCR will improve performance (especially sample throughput and dynamic range) while driving down costs for both digital PCR and digital MDA.
The ability to conduct contaminant-free MDA has important implications. For instance, positive and negative amplification results are more obviously apparent; small quantities of template DNA can be amplified with high fractional yield; and the reaction products can be analyzed without the false-positive signals or interfering sequences that arise from contaminants. In addition, there may be benefits of compartmentalization for certain studies that rely on MDA for preparative amplifications. MDA sequence and length bias may be reduced in metagenomic or library-based applications by eliminating competition among template molecules for amplification reagents. For example, a high-amplification-efficiency template molecule is limited to the reagents inside its microchamber, while a low-amplification-efficiency template molecule can be given extra time to catch up as it utilizes a privileged supply of reagents inside its own microchamber. Emulsion dMDA may prove to be another effective way to access the advantages of compartmentalized MDA.
Here, we introduced a new method, dMDA, for quantitatively enumerating the number of high molecular weight DNA fragments in a sample. We demonstrated the dMDA limit of quantification at 1
fg/ml for 2
kb fragments or less than one (fragmented) bacterial GE per microliter and identified a straightforward development path toward even better sensitivity. Digital MDA allows the enumeration of DNA fragments and other MDA-active DNA templates (such as plasmids and single- or double-stranded minicircles) on an absolute basis, without a standard curve. Furthermore, the digital nature of the microfluidic MDA results is consistent with high molecular weight DNA contaminants as the sole source of the so-called ‘template-independent’ MDA background. This method has applications in quality control of pharmaceuticals, biological and chemical reagents (especially DNA-modifying enzymes and assay components), as well as in characterization/quantification of DNA libraries, water quality testing, surveillance of industrial reactors, counter-bioterrorism, forensics, space exploration and astrobiology. Digital MDA is well-suited for analysis at the site of sample collection due to the low power requirements for isothermal incubation and straightforward interpretation of the raw data.
We also overproduced and purified a tagged ϕ29 DNAP with extremely low levels of nucleic acid contamination. Such a clean preparation of a strand-displacing polymerase is required to realize the low dMDA LOQ and LOD values quoted above. We characterized contamination in commercial lots of ϕ29 DNAP, finding levels sufficient to interfere with sensitive applications (such as single cell genomics or quantification of DNA fragments at concentrations <10
fg/ml) in every lot from the three vendors tested. High contaminant levels were also observed in most lots of MDA reaction buffers. We recommend that suppliers of reagents for ultrasensitive molecular biology applications use dMDA to quantify the concentration of contaminating DNA fragments and report the detected level in units of contaminating fragments per unit enzyme activity and fragments per reaction microliter to the end user. No contamination was detected above the dMDA LOD in the high-purity ϕ29 DNAP sample prepared in-house.