Name: simple bulk readout of digital nucleic acid quantification assays
Uploaded: 2015-09-24T15:00:00
Description: Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

The authors acknowledge Liyi Xu for providing MDA reagents and protocols. We also thank David Feldman and Navpreet Ranu for discussions relating to this work. This work was supported in part by a Burroughs Wellcome Career Award at the Scientific Interface to PCB.

Note: Fabricating the microfluidic device is not necessary for this assay, as droplets for this protocol can be formed with existing commercial droplet makers23,29.
1. Make the Droplet-forming Microfluidic Device
<ol>
	<li>Prepare master mold for the channels with the SU-8 Master Fabrication protocol outlined previously31, but with a droplet generator mask pattern32.</li>
	<li>Fabricate devices in PDMS using the techniques of soft lithography32,3...

<ol>
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Digital assays are powerful methods that enable detection of rare cells and counting of individual of nucleic acid molecules. However, digital assays are still not routinely applied in analytical laboratories, due in part to the cost of specialized equipment associated with commercially available methods. Here we describe an endpoint digital assay for quantifying nucleic acids with a simplified analog readout using a standard real-time quantitative PCR machine. This method is quick to set-up, and readout is much simpler ...

Digital assays for nucleic acid quantification (digital PCR)1-4 and base order (sequencing) are strongly impacting the life sciences and medicine. Digital assays provide quantification of molecular counts on an absolute scale (not relative to a control), providing high sensitivity, enabling facile comparisons across experiments, and crucially, enabling the construction of large databases containing comparable data5 (Table 1).
Over the last 15 years, whole genome amplification (WGA) emerged alongside PCR as a general tool for nucleic acid amplification. Like PCR, WGA is useful for analytical and preparative applications by amplifying minute sample elements up to a level that can be easily detected or used for subsequent analyses like base sequencing. Unlike PCR, WGA is not specific to a particular DNA locus, rather allowing amplification of all sequences in the sample, including unknown sequences. This fundamental difference between PCR and WGA makes the methods complementary to one another and gives rise to different challenges in their application.
The high yield of WGA reactions6 enables routine amplification of genomic DNA from single molecules7, single cells8 and other low-biomass samples9 for quantification or further analysis. The major challenges associated with WGA chemistry are its extreme sensitivity to contaminants and the uneven amplification across individual template molecules6. However, WGA is gaining popularity as single-cell sequencing has emerged as the &#8220;killer application&#8221; of WGA technology10, and template quantification by WGA is important in&#160;many fields of application7.
Instrumentation for digital nucleic acid quantification has been previously described in a variety of valved and valveless microfluidic formats11-14, including droplet-based assays15,16 (Table 2). However, commercial microfluidic systems for digital analysis require specialized equipment for reaction setup and product detection17. Custom valved microfluidics are flexible, but require precision microfabrication and pneumatic control systems18. While it is relatively simple to make monodisperse micro-droplets for emulsion-based digital assays19,20, digital readout is technically burdensome, requiring either large-scale wide-field imaging (similar to popular next-generation sequencing technologies)21,22 or high-speed flow-based droplet detection23-25. Ideally, a digital assay would be simple from set-up to readout, reducing the need for complex instrumentation and allowing large numbers of samples to be read out quickly. Here we describe a simplified method for readout of digital droplet assays that uses a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
While the new approach can be applied to digital PCR assays, it is particularly advantageous for digital WGA assays for which analog real-time amplification assays (that give excellent results for PCR) are problematic. WGA is commonly applied to samples with template molecules that are heterogeneous in sequence, length, and base content. These different template molecules are amplified at different rates6, necessitating the use of a reference (&#8220;standard&#8221;) sample with matching characteristics. Often, no such standard is available, or the characteristics of the incoming samples are unknown. The heterogeneity of the input material and length-dependent property of WGA chemistries26 also complicate the interpretation of results by creating ambiguity in what is being quantified--the input mass, input number of molecules, a combination of the two, or neither. Finally, the sequence-non-specificity renders quantitative multiple displacement amplification (MDA) more sensitive to contamination than quantitative PCR since contaminant molecules of any sequence have a potential to interfere. Microfluidic digital assays address contamination by segregating template molecules and reducing reaction volumes such that fewer contaminants are sampled.
Here we use a popular isothermal WGA method, MDA27. Of note, several other WGA chemistries including PicoPlex and MALBAC28 depend crucially on initial isothermal strand displacement steps. Isothermal steps exacerbate the challenge of applying analog real-time assays for quantitative WGA. WGA can neither be entirely prevented during setup, leading to unwanted variable pre-amplification, nor discretized (&#8220;cycled&#8221;) in a way where replication of heterogeneous molecules can be driven to completion and stopped prior to the next cycle like PCR11 (Figure 1). A digital assay format for WGA accomodates typical&#160;reaction setup procedures due to the segregation of each molecule&#160;for enumeration at the assay endpoint (so pre-amplification does not affect the results) original readout&#160;means accuracy would be largely independent of variation in amplification efficiency.
The assay depends on droplets produced in oil with uniform volumes, as the signal level per droplet at the assay endpoint will depend on the droplet volume, and we do not want the consistency of results to depend on averaging across a distribution of droplet sizes. Making monodisperse droplets is now a standard procedure (about 3,500 monodisperse droplet papers have been published since 2013), but requires microfluidic instrumentation25. In fact, more than six companies have developed independent commercial products that rely on production of such droplets, and droplet-making microfluidic chips are commercially available23,29. For this study, we used custom microfluidic devices produced in-house (see Protocol). Syringe pumps to drive flow through the devices are also commercially available, but alternatively, can be substituted with a single disposable syringe for vacuum-driven flow to reduce costs30.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Evagreen Dye</td>
			<td>Biotium</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>New England Biotechnologies</td>
			<td>B9000S</td>
			<td></td>
		</tr>
		<tr>
			<td>Phi29 DNA Polymerase Reaction Buffer</td>
			<td>New England Biotechnologies</td>
			<td>B0269S</td>
			<td>Vortex periodically until aggregate dissolves</td>
		</tr>
		<tr>
			<td>Lambda DNA</td>
			<td>New England Biotechnologies</td>
			<td>N3011S</td>
			<td>Heated to 50C and quenched on ice prior to dilution</td>
		</tr>
		<tr>
			<td>Randomized MDA oligos</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#39;-NNNNN*N-3&#39;</td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td>Integrated DNA Technologies</td>
			<td></td>
			<td>5&#8217;-CGGCAAACGGGAATGAAACGCC-3&#8217; and 5&#8217;-TGCGGCAAAGACAGCAACGG-3&#8217;</td>
		</tr>
		<tr>
			<td>UltraPure Nuclease-free water</td>
			<td>Life Technologies</td>
			<td>10977-015</td>
			<td>UV-treated for 30m in Stratalinker 2400 before use (optional)</td>
		</tr>
		<tr>
			<td>ROX reference dye</td>
			<td>Life Technologies</td>
			<td>12223-012</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR optical strip caps</td>
			<td>Life Technologies</td>
			<td>4323032</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Agilent Technologies</td>
			<td>401428</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP (25uM each)</td>
			<td>Agilent Technologies</td>
			<td>200415</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler - Mx3000P qPCR system</td>
			<td>Agilent Technologies</td>
			<td>401403</td>
			<td></td>
		</tr>
		<tr>
			<td>Barrier pipette tips</td>
			<td>VWR</td>
			<td>89003-046</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-rad oil for evagreen</td>
			<td>Bio-rad</td>
			<td>186-4005</td>
			<td></td>
		</tr>
		<tr>
			<td>JumpStart Taq ReadyMix</td>
			<td>Sigma-Aldrich</td>
			<td>P2893-100RXN</td>
			<td></td>
		</tr>
	</tbody>
</table>

simple bulk readout of digital nucleic acid quantification assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

While conventional bulk/real-time readouts can be used for both quantitative PCR and quantitative WGA assays (Figure 1), digital quantitative assays provide advantages (Table 1). In the method described, we read out digital assays in micro-droplet format with a simple bulk endpoint measurement (Figure 2). While this method is broadly applicable, we focus on quantitative WGA (MDA) because this method presents special challenges for conventional real-time assays.
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Watch this Scientific Journal Video about Simple Bulk Readout of Digital Nucleic Acid Quantification Assays at JoVE.com

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays (Video) | JoVE

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

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NanoDrop Microvolume Quantitation of Nucleic Acids

NanoDrop Microvolume Quantitation of Nucleic Acids (Video) | JoVE

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Electricity-Free, Sequential Nucleic Acid and Protein Isolation (Video) | JoVE

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Analyzing and Building Nucleic Acid Structures with 3DNA

Analyzing and Building Nucleic Acid Structures with 3DNA (Video) | JoVE

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

FtsZ Polymerization Assays: Simple Protocols and Considerations

FtsZ Polymerization Assays: Simple Protocols and Considerations (Video) | JoVE

During bacterial cell division, the essential protein FtsZ assembles in the middle of the cell to form the so-called Z-ring. FtsZ polymerizes into long ...

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays

Preparation, Imaging, and Quantification of Bacterial Surface Motility Assays (Video) | JoVE

Bacterial surface motility, such as swarming, is commonly examined in the laboratory using plate assays that necessitate specific concentrations of agar ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization (Video) | JoVE

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method (Video) | JoVE

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model (Video) | JoVE

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

The Lactate Dehydrogenase Sequestration Assay Ã¢â‚¬â€ A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells (Video) | JoVE

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...