Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Summary

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.

Abstract

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 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.

Introduction

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 prepara....

Protocol

Note: Fabricating the microfluidic device is not necessary for this assay, as droplets for this protocol can be formed with existing commercial droplet makers^23,29.

1. Make the Droplet-forming Microfluidic Device

1. Prepare master mold for the channels with the SU-8 Master Fabrication protocol outlined previously³¹, but with a droplet generator mask pattern³².
2. Fabricate devices in PDMS using the techniques of soft lithography^32,33.

2. Prepare Reaction Mix for Bulk Droplet Readout

Note: The protocol can b....

Results

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.

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Evagreen Dye	Biotium	31000
Bovine serum albumin	New England Biotechnologies	B9000S
Phi29 DNA Polymerase Reaction Buffer	New England Biotechnologies	B0269S	Vortex periodically until aggregate dissolves
Lambda DNA	New England Biotechnologies	N3011S	Heated to 50 oC and quenched on ice prior to dilution
Randomized MDA oligos	Integrated DNA Technologies		5'-NNNNN*N-3'
PCR primers	Integrated DNA Technologies		5’-CGGCAAACGGGAATGAAACGCC-3’ and 5’-TGCGGCAAAGACAGCAACGG-3’
UltraPure Nuclease-free water	Life Technologies	10977-015	UV-treated for 30 min in Stratalinker 2400 before use (optional)
ROX reference dye	Life Technologies	12223-012
PCR optical strip caps	Life Technologies	4323032
PCR tubes	Agilent Technologies	401428
dNTP (25 micromolar each)	Agilent Technologies	200415
Thermocycler - Mx3000P qPCR system	Agilent Technologies	401403
Barrier pipette tips	VWR	89003-046
Bio-rad oil for evagreen	Bio-rad	186-4005
JumpStart Taq ReadyMix	Sigma-Aldrich	P2893-100RXN