Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

The overall goal of this procedure is to simplify readout of digital droplet assays using a conventional realtime PCR instrument to measure bulk fluorescence of the assay. This is accomplished by first preparing amplification reactions of the samples of interest, two standards and forming droplets to partition each into thousands of nanoliter sized reactors. The second step is to incubate the droplets to amplify the nucleic acids within each droplet.

Next, the bulk fluorescence of each sample or standard is measured with a standard QPC or thermocycler. The final step is to predict the fraction of fluorescent droplets in the samples of interest using a standard curve generated from the standard samples. To verify the performance of this method, fluorescent microscopy can be used to independently evaluate the quantification performance of the bulk fluorescence assay.

The main advantage of this technique over existing methods like droplet counting, is that it does not require specialized instrumentation Prior to starting this assay repair. All necessary reagents as described in the protocol text two standards are required. One, no template control, such as nuclease free water and one high template concentration such as lambda, DNA, denature 3.3 microliters of each standard with 3.3 microliters of denaturation buffer in A-Q-P-C-R tube.

Incubate it room temperature for three minutes. Quench each with 3.3 microliters of neutralization buffer. Similarly, denature 3.3 microliters of the template of interest with 3.3 microliters of denaturation buffer incubated room temperature for three minutes.

Quench with 3.3 microliters of neutralization buffer. Prepare 11 microliters of master mixture per sample for fewer or false positives. Expose the master mix to UV light prior to forming droplets.

First, prepare 10 microliters of premix mixture per sample in a 0.5 milliliter or 1.5 milliliter clear tube on ice. The premix mixture consists of randomized oligo, B-S-A-D-N-A polymerase reaction buffer, dn, NTPs, and nuclease free water. Place the tube in water on ice and expose to UV light after the UV exposure.

A-D-S-D-N-A binding dye and reference diet to the premix mixture. Add PHI 29 DNA polymerase last to ensure the polymerase does not encounter pH levels much higher or lower than 7.5. Mix well combine 10 microliters of denatured template or standard and 10 microliters of the master mixture mix well.

Since droplets can be very sensitive to static electricity and sheer stress, the experimenter should remove clothes that may cause static electricity and ground herself in place before forming droplets. The microfluidic device used for this demonstration was produced in-house and has an oil inlet and two aqueous inlets with a flow focusing junction for generating droplets. Use two syringe pumps to control the flow rates of 55 microliters of oil with surfactant and 20 microliters of reaction mixture through the microfluidic chip.

To generate 20, 001 nanoliter droplets, One of the most difficult steps is collecting droplets without disturbing the emulsion. It's important to pipette slowly and preferably. Use a wide board tip.

Collect all of the droplets into PCR tubes for each sample. Aliquot 30 microliters of oil into fresh PCR tubes with optical caps using a wide bore tip and pipetting very slowly. Transfer 20 microliters of droplets on top of the oil.

The consistent volumes ensure the assay is as accurate as possible. Close the tube caps tightly as loose caps will allow the oil to evaporate handle tubes of emulsion from the top of the tube as far from the emulsion as possible. A PCR thermocycler, A-Q-P-C-R thermocycler or a hot plate can be used for isothermal amplification.

A-Q-P-C-R thermocycler is used in this demonstration. Incubate the samples for seven hours at 30 degrees Celsius and inactivate for one minute at 75 degrees Celsius. Immediately following reaction and activation.

Measure di fluorescence levels for all samples using the QPCR thermocycler. For each sample. Divide the fluorescence intensity of the D DS DNA binding dye by the fluorescence intensity of the reference dye.

Subtract the background fluorescence or the normalized fluorescence of the no template control from all samples. Create a linear standard curve using the corrective fluorescence measurements from the standards. The no template control standard represents the fluorescence for a sample with 0%fluorescent droplets and the high template concentration.

Fluorescence measurement is the expected fluorescence for a sample with 100%fluorescent droplets using the corresponding standard curve. Predict the intermediate ratios of positive and negative droplets based on their bulk fluorescence. This method uses a conventional real-time PCR instrument to measure bulk fluorescence of droplet based digital assays.

Fluorescent and non fluorescent droplets were pre-mixed in different ratios and a comparison made between bulk fluorescence and the fraction of positive droplets. As expected, the bulk fluorescence and positive droplet counts scale linearly with the input fraction of fluorescent droplets. Predicted ratios based on standards were compared to the expected fluorescent input fractions.

The line indicates the expected value given a linear relationship. The results show good performance in droplet ratio quantification across two logs of dynamic range. To test this method in a real quantitative whole genome amplification assay, fluorescence levels, and fractions of positive droplets of a digital droplet.

MDA assay with increasing concentrations of template Lambda DNA were measured. The line indicates the expected fluorescent fraction using the poissant distribution to model the data from the experiment. Representative fluorescent images of the droplets are shown both bulk fluorescent and positive droplet fraction from the digital MDA samples scale as expected with the average template per droplet, indicating that the bulk readout can faithfully capture the result of a digital asay.

This method can benefit other digital assays like digital PCR by speeding up parallelizing and simplifying assay readout.