Tutorial Level

# Drop-off Assay for Determining Mutant DNA Population

Digital PCR (dPCR) can now be used for a wide range of applications and offers superior precision to traditionally used qPCR. Several digital PCR applications robustly and reliably detect sequence mutations and are particularly advantageous in an oncology context. For example, as described in the Rare Mutation Detection tutorial, dPCR can detect rare mutations in samples with a low concentration of the target DNA.

Importantly, using a drop-off assay, dPCR can maximize the data output of samples in a single assay by simultaneously detecting the presence of multiple sequence insertions, deletions or mutations occurring within a defined genomic interval.

In this tutorial, we will walk you through a drop-off assay, from setting it up to analyzing the data. Here, we use the example of detecting a set of the most prevalent KRAS mutations on Exon 12 (KRAS G12) in colorectal cancer.

Other important applications for a Drop-off assay include detection of BRAF, NRAS, EGFR and other mutations in oncology. Additionally, a Drop-off assay can also be used to detect the mutations induced using CRISPR.

## 1. How does a drop-off assay work?

A drop-off assay uses two probes: one wild-type (WT) or drop-off probe and one reference probe. The WT probe spans the mutation hotspot but is uniquely complementary to the wild-type sequence, whereas the reference probe binds specifically to the WT allele in a region adjacent to but nonoverlapping with the mutation site (Figure 1). In the case of a WT allele, both the probes bind to their complementary regions and produce a fluorescent signal, hence producing a double positive population. In the case of a mutation/deletion/insertion, the binding of the WT probe is destabilized and no longer recognizes its target, hence yielding only a single positive population for the reference probe.

## 2. How to design a drop-off assay?

Note: The rules for designing primers and probes for dPCR and qPCR follow similar principles.

In this example assay, we use one set of primers: the KRAS WT probe labeled with the Cy5 fluorophore, and the KRAS reference probe labeled with the FAM fluorophore.

Figure 1. Illustration of hybridization positions of the WT and Reference probes and the primer pair (arrows) on the amplified target (FP = forward primer; RP = reverse primer).

## 3. What are the materials required for this assay?

The materials required for a dPCR experiment are:

• dPCR equipment with the capacity to simultaneously detect at least two different fluorescence wavelengths
• PCR MasterMix (standard kit) containing the required reagents including:
• DNA polymerase
• Deoxyribonucleotides
• Reaction buffer
• Magnesium chloride
• Nuclease-free water
• Fluorescein (a reference dye; varies from different manufacturers for the specific kits)

Specifically, for this experiment, the following are required:

• WT DNA
• Sample DNA containing the KRAS G12 mutated sequence
• Forward and reverse primers to amplify KRAS DNA (see Table 1)
• A Cy5 labeled hydrolysis probe to detect the WT KRAS sequence (see Table 1)
• A FAM-labeled hydrolysis probe to detect the KRAS reference sequence (see Table 1)
• The restriction enzyme Tru1L

Important note for a drop-off assay in oncology:

1. The desired amplicon size in a drop-off dPCR reaction should be <120bp. Indeed, DNA originating from patient liquid biopsy samples (circulating tumor DNA or ctDNA) and formalin-fixed, paraffin-embedded (FFPE) samples are frequently used as a template source for dPCR and are fragmented due to the process of natural cell death or chemical treatment, respectively.
2. In the case where high molecular weight DNA or unfragmented DNA samples are used, using a restriction enzyme or sonicating the samples to an average size of about 200bp is highly recommended to fragment the DNA for optimal population separability. However, it is mandatory to verify that the chosen restriction enzyme does not target any of the amplicons. For our experiments, we used Tru1L after confirming efficiency in our samples.
3. Adding chemical modifications such as a Minor Grove Binder (MGB) and Locked-Nucleic Acid (LNA) bases to the probes helps to increase their melting temperature (TM) and, as such, their target specificity.

## Digital PCR Principle

Since the invention of PCR by Kary Mullis in 1983, it has been a real revolution in molecular biology. A decade later, real-time PCR also termed quantitative PCR, offered the possibility of monitoring the PCR process. Until now, real-time PCR is considered as the “gold standard” for gene detection and quantification. But real-time PCR has its drawbacks such as the need for an external reference for quantification and a lack of sensitivity at low concentrations.

To further assess these drawbacks, Digital PCR was developed. The aim was to enhance the sensitivity of real-time PCR, especially in cases where detecting point mutations of low abundance in a background of wild-type DNA is required.

This technology is based on PCR amplification of DNA templates that have been randomly isolated into individual reactions. Each reaction contains zero, one or more molecules.

PCR amplification is then conducted using fluorescence dyes or probes, similar to real-time PCR.

The number of positive and negative reactions is counted by assessing the fluorescence intensity of each reaction at the end of the PCR reaction. For each reaction, a binary result is obtained explaining the term “digital”.

Finally, the proportion of positive and negative reactions is fitted into a Poisson distribution in order to obtain the absolute concentration of target molecules, eliminating the need for external references. Learn more about Poisson Distribution in Digital PCR in this detailed item: Poisson Law Computation.

A free calculator that automatically estimates target gene concentrations is also available here: Poisson Law: going further

The key feature of digital PCR is the compartmentalization of the sample. This isolates the DNA fragments from each other and then creates an artificial enrichment of low abundance sequences, leading to a higher detection level.

Finally, the dynamic range of the method is dependent on the total number of compartments generated.

### Technologies to achieve a digital PCR

Until recently, digital PCR was performed using PCR plates. This process was labor-intensive and reagent consuming- limiting its use for routine analysis.

The latest advances in microfluidics enable massive automated partitioning of DNA sample in hundreds to millions of nano- or pico-liter scale compartments. Digital PCR array chips, splitting the sample in hundreds of microfluidic wells, combined with real-time PCR machines was the first digital platform developed.

An even higher throughput partitioning was obtained with a water-in-oil emulsion. Using microfluidic circuits and surfactant chemistry, thousands to millions of monodispersed partitions containing template DNA and PCR reagents can be generated, thermocycled, and then analyzed using automated partition flow-cytometers or scanners.

### Digital PCR Instruments

Several digital PCR platforms exist on the market. They propose different technologies to achieve sample partitioning, thermal cycling, and digital readout.

Here is a list of links to the websites of digital PCR providers where you will find the information needed to achieve digital PCR experiments according to their standards:

### Applications of Digital PCR

Digital PCR is constantly proving its reliability in fields of application where precision and sensitivity are vital.

Thanks to its exquisite sensitivity, mainly required for the detection of point mutations, digital PCR met its first application in the field of oncology.

Wider applications in clinics and human biology were then developed including prenatal diagnosis, organ transplantation, and diabetes monitoring.

Digital PCR has proven its adaptability in numerous molecular assays and is now recognized as a reliable method not only for rare alleles and rare mutations detection but also for genotyping, absolute quantification, and copy number variation.

The areas of research where dPCR can be utilized are rapidly increasing. dPCR has been successfully implemented in the fields of virology, food and environmental microbiology applications, and for the monitoring of genetically modified organisms.

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## PCR Basic Knowledge

“PCR” stands for Polymerase Chain Reaction.

PCR was first developed in 1983 by Kary Mullis, who was awarded the Nobel Prize in 1993 for this extraordinary invention. Indeed, PCR enables the clonal amplification of specific segments of DNA from a single fragment to thousand of millions of copies!

This tool is widely used in molecular biology for a variety of applications- manipulating DNA (cloning, mutagenesis), analyzing genes (though PCR-based sequencing for example), diagnosis or monitoring of pathologies, and also for parental testing or for DNA profiling in forensic studies.

### 1. What is in a PCR mix?

• #### Polymerase

First and foremost, a PCR mix contains a thermostable polymerase, such as the ubiquitous Taq. The Taq polymerase was originally isolated from the thermophilic bacterium Thermus aquaticus in 1971 and is the original enzyme that was used for PCR by Kary Mullis. Since then, many other polymerases have been discovered. They have different properties regarding their tolerance to inhibitors, processivity (length of amplicon generated), speed of synthesis, etc. However, the Taq polymerase, whether in its original configuration or incorporating engineered elements, remains a favorite for PCR-based amplification.

• #### dNTPs

The nucleotides are the building blocks of the reaction. They are present in the form of deoxyribonucleotides and are commonly referred to as dNTPs. The “dNTP” denomination means that dCTP (deoxycytidine triphosphate), dATP (deoxyadenosine triphosphate), dTTP (deoxythymidine triphosphate), and dGTP (deoxyguanosine triphosphate) are present in equimolar amounts. Depending on the application, the concentration of individual nucleotides may be further tuned, or some nucleotides may altogether be substituted with analogs, such as 5-fluorouracil or 6-mercaptopurine.

• #### Reaction Buffer

Although the polymerase may be active over several pH points, the pH of most PCR commercial mixes is usually around 8 – 8.4 for optimal amplification. The pH of the reaction buffer is controlled by the use of a buffering agent, most commonly Tris-HCl. The reaction also comprises monovalent cations such as K+ and NH4+, divalent cations such as Mg2+, and less commonly Mn2+. The K+ salts neutralize the negative charge on the backbone of DNA and thus stabilize primer-template binding. Mg2+ is an essential co-factor of the polymerase. Its concentration in the reaction affects the specificity and efficiency of the reaction.

Other chemical species can be found in PCR mixes, such as detergents, additional salts, DMSO, or proteins such as bovine serum albumin. These compounds can be included to help with denaturation of the DNA strands, especially in sequences with high GC content, to improve polymerase activity, or to counter the inhibitory effects of substances present in the samples.

• #### Primers

In order to amplify a DNA fragment by PCR, two DNA primers (single-stranded oligonucleotides) that are complementary to the 3’ ends of the sense and anti-sense strands of the DNA target are needed (Figure A). To know more about primers, refer to the “Designing Primers and Probes” item!

Figure A. Primers anneal to their target on each strand of DNA during PCR and yield amplicons defined by the hybridization location of each primer on the template.

• #### Template DNA

Template DNA can be any DNA to be amplified, whether extracted from animal or plant cells, fungi, bacteria, or present in free form- for example circulating DNA in blood or urine. For optimal results, it is recommended to use long-enough purified DNA fragments for amplification to take place. Additionally, caution needs to be exercised to avoid chemical and/or UV-induced damage to the DNA template as it may affect your PCR results.

### 2. Thermal Cycling in PCR

PCR is based on thermal cycling: the reaction mix is subjected to sequential cycles of heating and cooling, which allow specific temperature-dependent reactions to occur.

• #### Denaturation

First, DNA is denatured or melted at a high temperature, typically 94-96°C. During this step, the hydrogen bonds between complementary bases of the double-stranded DNA are broken, yielding two single-stranded molecules. Depending on the type of DNA, the reagents, and equipment used this step usually lasts ranging from 15 seconds to 1 minute and marks the start of each cycle of PCR.

• #### Annealing

After denaturation of the template DNA strands, primers anneal to their target on the DNA template. The temperature of this step is critical to ensure specific hybridization of the primers to the target. In practice, this temperature is usually determined empirically- it needs to be low enough to allow primer binding but high enough for the binding to be specific to the intended target. Usually, annealing temperatures are between 3-5°C below the calculated Tm of primers, which translates to annealing temperatures for PCR between 50-65°C. Once the primer has annealed to the DNA, the polymerase binds to the primer-template hybrid and elongation can start.

• #### Elongation

During elongation, the polymerase synthesizes new DNA strands by adding free nucleotides (dNTPs) present in the PCR reaction one-by-one, and the polymerization of the new strand complementary to the template takes place in the 5’ to 3’ direction

The temperature for elongation depends on the polymerase used, with 68-72°C being most commonly used. The length of the amplicon is taken into account when determining the time needed for the elongation step with a general rule of thumb of 1 minute per kb to be synthesized. The synthesis rate of the polymerase present in your PCR mix can be found in the manufacturer’s recommendation.

Initial denaturation

Before cycling starts, a longer initial denaturation step is often included (90-95°C for 1-10 minutes). Why? Because hot-start polymerases must be activated as these enzymes have been developed to reduce non-specific amplification early in the PCR reaction. Thus, they are locked in an inactive state by binding to an antibody or a chemical inhibitor. At elevated temperatures, a non-reversible dissociation occurs and this unlocks the enzyme.

Final Elongation

A final extension step  (68-72°C for 5-10 minutes) can also be included to ensure all amplicons are fully elongated.

### 3. What happens during PCR?

• #### Number of copies of target DNA fragment generated during PCR

If the reaction efficiency is 100%, the number of amplicons doubles after each cycle. This means that each copy of the DNA target initially present will be amplified 2n, where n is the number of cycles of PCR. Thus, when amplifying a single copy of your target region for 40 cycles, you could finally get 240 = 1.1 x 1012 amplicons!

• #### Stages of PCR

Given that the number of amplicons doubles at each cycle when PCR efficiency is 100%, PCR amplification is referred to as exponential.

However, since this is an enzymatic reaction one or more factors will become limiting as the reaction progresses. Moreover, the polymerase may also start to lose activity. This will mean that at some point the amplification will start to level off, before reaching a plateau where no more products are generated.

### 4. How are the PCR results visualized?

• #### Electrophoresis

Traditionally, the products of PCR are run on an agarose gel to visualize the amplicons. The agarose gel is stained with an agent that will bind to DNA (ethidium bromide or GelRed® for example) and illuminated with UV light in order to reveal the various DNA fragments present. Typically, a DNA ladder is run alongside the PCR products to estimate the size of the products, as well as the amount of DNA generated (Figure B).

Other electrophoresis techniques may use capillary gels and fluorescently labeled primers for fine discrimination of amplicon size.

Figure B. Agarose gel electrophoresis. Lane 1: DNA ladder, Lane 2: PCR product 1, Lane 3: PCR product 2

• #### Fluorescence

Different chemistries allow the fluorescence detection of DNA molecules. Amongst these chemistries, Hydrolysis probes, SYBR® Green I, and EvagGeen® are the most commonly used.

Hydrolysis probes (TaqMan® probes)

A probe, like primers, corresponds to a short oligonucleotide sequence (different from primer sequences) complementary to the target DNA sequence. Located between the sense and anti-sense primers, the hydrolysis probe consists of a fluorescent dye and a quencher. During amplification, the hydrolysis probe will be cleaved by the 3’->5’ exonuclease activity of the Taq polymerase. This will separate the fluorescent moiety from its quencher (Figure C). These fluorescent molecules will accumulate after each cycle. A fluorescent signal proportional to the number of amplicons bearing the probe hybridization sequence can be recorded. If you want to know more about probes, refer to the “Designing Primers and Probes” item.

Figure C. Hydrolysis probes. 1. The probe is a single-stranded DNA sequence labeled with a fluorophore at its 5’ end and a quencher at its 3’ end. The fluorescence of the fluorophore is quenched, i.e., absorbed by the quencher- no light is emitted. 2. During PCR, the probe will hybridize with the amplicon generated in a sequence-specific manner. 3. The 3’ to 5’ exonuclease activity of the Taq polymerase destroys the hybridized DNA probe, thus freeing the fluorophore from the quencher. This fluorophore is now free to emit a fluorescent signal.

DNA-binding dye: EvaGreen®

EvaGreen® is a DNA binding dye that can be used to track PCR amplification in most digital PCR systems. It is a green fluorescent dye which becomes highly fluorescent when it is bound to double-stranded DNA. EvaGreen® is a thermoresistant molecule with excitation and emission spectra close to that of SYBR® Green I  or 6-FAM dyes. Please note that the advantage of EvaGreen® over SYBR® Green I is its compatibility with droplet-based digital PCR systems. Thus, SYBR® Green I is mostly used in real-time PCR.

When using EvaGreen, the intensity of the fluorescent signal will correlate with the length of the amplicon generated. The longer the amplicon, the more bases available for EvaGreen binding, thus the stronger the fluorescence signal (Figure E).

Figure D. Excitation and emission spectra of EvaGreen® in the presence of double-strand DNA in PBS buffer

Figure E. EvaGreen® dye binds to double-strand DNA via a “release-on-demand” mechanism.

[https://biotium.com/technology/pcr-dna-amplification/evagreen-dye-for-qpcr/]

Fluorescence detection is the detection system used both in digital and real-time PCR.

Further information about EvaGreen® can be found on the manufacturer’s website

#### Digital PCR

Most digital PCR platforms use end-point PCR for quantification. End-point PCR means that the results of PCR are assessed when the plateau is reached. It occurs when the amplification has stopped due to loss of polymerase activity or an essential reagent (primers for example) present in the reaction.

In these reactions, fluorescent reporters are used. It can be DNA-binding dyes such as EvaGreen or fluorescently-labeled hydrolysis probes.

For further information on digital PCR refer to the “Principle of Digital PCR” item.

#### Real-time PCR

The intensity of the fluorescent signal is recorded at each cycle of PCR. The results are visualized on a graph where the intensity of fluorescence is plotted against the number of cycles. During the first few cycles, the fluorescent signal generated by the fluorescent reporter is indistinguishable from the background. As amplification takes place during cycles, the fluorescence eventually increases above noise level before tailing off and reaching a plateau.

Due to the accumulation of amplicons, the exponential increase of fluorescence can be visualized on the graph. A threshold for fluorescence intensity is placed at the beginning of this exponential phase. The cycle number at which the amplification curve intersects the threshold is called Cq (Cycle quantification). The quantification of an unknown sample assumes the use of a standard curve. By comparing the Cq obtained for DNA samples of known concentration with the Cq obtained for the unknown sample,  DNA concentration in the unknown sample is then determined (Figure F)

Figure F. Real-time PCR results. Fluorescence intensity is recorded at every cycle and plotted against the cycle number. Reaction 1 has a known DNA concentration (Cq1). At 100% efficiency, the DNA content in the reaction doubles at each cycle. Broadly, if Cq2 is distant from Cq1 from 1 cycle, this means there was half as much DNA in reaction 2 as there was in reaction 1 at the start of the PCR.

## Designing Primers & Probes

Primer and probe design is a crucial step for a successful experiment.

The rules for designing primers and probes in a digital PCR assay are similar as for a qPCR assay:

### 1. Primers

Their length should be between 18 and 25 base pairs.

The criterion that you have to carefully monitor are:

• Percentage of GC: 40-60%
• Primer melting temperature (Tm): ideally between 55-65°C and Tm between both primers should not differ by more than 5°C
• G or C bases at the 3′ of the primer but not more than 2 in the last 5 bases
• Low probability of dimer or hairpin formation

### 2. Probes

The length should not exceed 30 base pairs. Ideally 15 base pairs for optimal specificity.

The criterion that you have to carefully monitor are:

• Percentage of GC: 20% to 80%
• Probe melting temperature: ideally from 4 to 10°C above the primer melting temperature
• Absence of more than 4 G repeats
• Low probability of dimer or hairpin formation
• Absence of G at the beginning of the probe

Sometimes, you will need to increase the Tm of the probe while keeping its size short. You can use modified bases such as locked nucleic acid (LNA) bases or peptide nucleic acid (PNA) bases. A probe with a minor groove binder (MGB) group is also an option to increase the Tm.

For further information about probe design, please refer to the following publications:

• Design of primers and probes for quantitative real-time PCR methods. Rodríguez A, Rodríguez M, Córdoba JJ, Andrade MJ, Methods Mol Biol. 2015;1275:31-56. doi: 10.1007/978-1-4939-2365-6_3. [PMID: 25697650]
• http://media.axon.es/pdf/78099_2.pdf

• Locked nucleic acids in PCR primers increase sensitivity and performance. Ballantyne KN, van Oorschot RA, Mitchell RJ. Genomics. 2008 Mar;91(3):301-5. doi: 10.1016/j.ygeno.2007.10.016.[PMID: 18164179]
• An introduction to peptide nucleic acid. Nielsen PE, Egholm M. Curr Issues Mol Biol. 1999;1(1-2):89-104. Review. [PMID: 11475704]

Tools for primer and probe designs:

## 4. Preparation of the PCR mix

Note: This protocol has been optimized using the Naica™ System and the Sapphire Chip. The Naica System has the capacity to detect targets in 3 colors. The ability to detect and analyze the red channel (for example the Cy5 fluorophore) is unique to the 3-color Naica System. In this tutorial, the FAM hydrolysis with the reference sequence was detected in the blue channel, and the Cy5 hydrolysis upon hybridization with the WT sequence was detected in the red channel. An additional channel (green; HEX, Cy3) can be used to detect a target or internal control.

Why is it critical to purify the DNA and quantify it? Follow this link to learn how you can purify and perform DNA preparation for Digital PCR.

It is also important to have a worksheet ready before sitting at the bench to perform an experiment, so you have all the calculations handy.

The calculations for digital PCR are similar to those for qPCR.

For each dPCR experiment, you need to know:

• The number of total samples to be analyzed in order to prepare a sufficient quantity of MasterMix: Always calculate for “n+1” samples, where “n” is the number of samples to be tested. For example, if you have 9 samples, prepare a MasterMix for 10 samples to have excess in case of any volume loss during pipetting.
• The instructions for the PCR mix:

## 5. What are the next steps to get my results?

#### a. Performing the experiment

Working in a clean, DNase-free space and having no sources of contamination is essential. A PCR hood is recommended. Mix the desired quantity of reagents based on the working sheet in a single tube before loading. The workflow varies for each dPCR equipment. Please follow the User Manual for the equipment you are using. For a brief overview- see this item about Performing Digital PCR Hands-on.

Thermal Cycling conditions used for this experiment:

Note that the thermal cycling conditions are assay dependent.

## Digital PCR DNA Preparation

DNA preparation is a crucial step for the success of your digital PCR assay. Template extraction, its quality control, and its storage are important parameters to monitor.

• ### Template extraction

Digital PCR has been reported to be compatible with DNA & RNA templates extracted by various extraction methods used in laboratory- from phenol chloroform method to modern extraction kits1,2,3,4,5,6

• ### Quality control

For optimal digital PCR performance, you should check the purity and the quality of your template. Using relevant methods such as spectrophotometry, you will be able to verify the good absorbance (A260/230 and A230/260) ratios of your extracted DNA solution. The presence of contaminants and/or potential inhibitors should be avoided.

• ### Storage

The storage of your extracted DNA template is also an important factor  to monitor. Indeed, for example, you need to avoid base degradation such as cytosine deamination and 8-Oxo-2′-deoxyguanosine formation due to oxidative damage since it may lead to base transversion during PCR amplification.

Buffering and temperature conditions have an impact on the quality of your sample. Storing at -20°C in Tris-EDTA is the standard condition, but please do not hesitate to take a look at the extraction kit manufacturer’s recommendations.

Other parameters, inherent to the sample itself, might have an impact on your digital PCR experiment and should be verified :

• ### DNA with high GC content

If your template contains a GC-rich region, this might conduct to an incomplete amplification. Indeed, GC bonds are highly stable. Thus, in order to obtain a better denaturation of your template, you can try to add DMSO or betaine in your PCR mix.

• ### High molecular weight DNA

High molecular weight DNA or plasmids have been reported by some manufacturers as problematic for partition generation.  Thus, we recommend DNA shearing using chemicals or enzymatic methods prior to performing digital PCR. Please note that the digestion step might be performed directly in the PCR mix.

Finally, before starting your digital PCR assay you will have to proceed on the :

• ### Conversion of mass of DNA in number of copies

If you are a qPCR user, you may be used to thinking in terms of mass of DNA input.

In digital PCR, you will need to think in terms of the number of copies of genes or genomes in your reaction volume.

In order to proceed on the conversion, the only thing you will have to know is the mass of the studied genome.

Then, simply apply this formula: Number of copies in reaction volume = mass of DNA in reaction volume (in ng)/ mass of the studied genome (in ng)

For more details about how to perform this calculation, refer to Part 3a of Rare Mutation Detection Tutorial.

Online calculators are also available :

### Bibliography

1 Cai, Y., Li, X., Lv, R., Yang, J., Li, J., He, Y., & Pan, L. Quantitative Analysis of Pork and Chicken Products by Droplet Digital PCR. BioMed Research International, 2014, 810209. http://doi.org/10.1155/2014/810209. PMID: 25243184

2 Pérez-Barrios, C., Nieto-Alcolado, I., Torrente, M., Jiménez-Sánchez, C., Calvo, V., Gutierrez-Sanz, L., Palka, M., Donoso-Navarro, E., Provencio, M., Romero, A. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: impact on biomarker testing.  Transl Lung Cancer Res. 2016 Dec; 5(6):665-672. doi: 10.21037/tlcr.2016.12.03. PMID: 28149760

3 Demeke, T., Malabanan, J., Holigroski, M., Eng, M. Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR. J AOAC Int. 2017 Mar 1;100(2):492-498. doi: 10.5740/jaoacint.16-0284. Epub 2016 Dec 22. PMID: 28118137

4 Holmberg, R.C., Gindlesperger, A., Stokes, T., Lopez, D., Hyman, L., Freed, M., Belgrader, P., Harvey, J., Li, Z. Akonni TruTip® and Qiagen® Methods for Extraction of Fetal Circulating DNA-Evaluation by Real-Time and Digital PCR. PLoS One. 2013 Aug 6;8(8):e73068. doi: 10.1371/journal.pone.0073068. Print 2013. PMID: 23936545.

5 Rajasekaran, N., Oh, M. R., Kim, S.-S., Kim, S. E., Kim, Y. D., Choi, H.-J., Byum, B., Shin, Y. K. Employing Digital Droplet PCR to Detect BRAF V600E Mutations in Formalin-fixed Paraffin-embedded Reference Standard Cell Lines. Journal of Visualized Experiments : JoVE, 2015, (104), 53190. Advance online publication. http://doi.org/10.3791/53190. PMID: 26484710.

6 Devonshire, A. S., Whale, A. S., Gutteridge, A., Jones, G., Cowen, S., Foy, C. A., & Huggett, J. F. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification. Analytical and Bioanalytical Chemistry, 2014, 406(26), 6499–6512. http://doi.org/10.1007/s00216-014-7835-3. PMID: 24853859

## Digital PCR Assay Optimization

Sub-optimal digital PCR settings very often lead to insufficient signal to noise ratio. This affects the separation between the negative and the positive partitions and prevents for an adequate threshold setting. That leads to limit the accuracy of the quantification and the sensitivity of the assay.

When developing an assay on a digital PCR system, there are several elements to consider:

• DNA template for assay optimization (positive control)
• Difference in fluorescence amplitude between negative and positive populations
• Presence of partitions of intermediate fluorescence (rain)
• Specificity – Are there non-specific populations? Can you detect positive partitions in your negative control?

### 1. DNA template for assay optimization

Ideally, the DNA template should be in the same form as it will be in the sample you will want to assay :  sheared in small fragments if targeting circulating cell-free DNA or relatively intact if assaying genomic DNA. The DNA solution used should be devoid of contaminants and potential inhibitors and have good absorbance (A260/230 and A230/260) ratios. If there is no evident source for DNA material to develop your assay, you may use synthetic oligos as templates for optimization.

• #### Presence of false positives

False positives are also a factor that contributes to the diminution of the sensitivity of the digital PCR assay. To limit this phenomenon, it is crucial to prevent DNA contamination in the laboratory or cross-contamination from well to well. Template quality is also a factor that is important to monitor as base degradation such as cytosine deamination and 8-Oxo-2′-deoxyguanosine formation due to oxidative damage may lead to base transversion during PCR amplification.

For more information about DNA preparation,  have a look at the “DNA Preparation For Digital PCR” item.

### 2. Difference in fluorescence amplitude between negative and positive populations

When you run an assay for the first time, you might not distinguish the negative and positive populations on a digital PCR system (Figure A).

Figure A. The positive partition population (blue) is barely separated from the negative population (dark grey), rendering thresholding difficult on this 1D dot plot. B. The fluorescence of the positive population (blue) is clearly separated from the negative population (dark grey), placing a threshold is therefore unambiguous.

Here are a few tips to improve the separability of your populations:

• Check manufacturers recommendations on primers and probe concentrations to be used in your digital PCR system. These may be higher than the ones usually recommended in qPCR.
• Optimize hybridization temperature. Check for the highest hybridization temperature that gives you an optimal separability and no rain (see below).
• Check the type of probes you are using. Double-quenched probes will give you a lower basal fluorescence signal, and therefore a higher separability.
• Check whether your probes are too old! Depending on how long your probes have been stored, whether they have been stored properly, or whether they just encountered too many freeze-thaw cycles, it is possible that your probes are already hydrolyzed. In this case, you may see a high fluorescence basal level, and, since there are only a few intact probes for PCR, a low signal for the positive partitions.
• If using a system where you can vary acquisition parameters, you may also try to tune the exposure time

### 3. Presence of partitions of intermediate fluorescence (rain)

We call “rain” partitions that have intermediate fluorescence values between the positive and the negative populations. In these partitions, amplification/hybridization efficiency is sub-optimal. The rain makes the threshold setting harder, and thus may affect quantification.

Various factors may be at the origin of the “rain” such as, template degradation, PCR inhibitors, polymerase errors, primer dimer, target accessibility (Figure B) or non-homogenous distribution of fluorescence into partitions.

Figure B. 1D dot-plots of a digital PCR experiment in partition using one fluorescent assay targeting a non-digested plasmid (A and B) and a digested plasmid (C). Rain is clearly visible on the two first plot whereas plasmid digestion has removed most of the partitions corresponding to rain.

In order to minimize the rain, you can optimize your digital PCR reaction by:

• Checking the optimal hybridization temperature as described above
• Checking your DNA template for anything that may prevent accessibility to the target. Is this a GC-rich region? Try additives such as DMSO or betaine. When using high molecular-weight DNA or plasmids, it is also best to fragment the DNA by digestion or mechanical means prior to digital PCR. If using digestion, you may also be able to perform this step directly in the mix.
• Making sure to use DNA free from inhibitors
• Increasing the number of cycles in order to ensure that all partitions reach the reaction plateau.

### 4. Presence of non-expected populations

When assaying a single target, you usually expect a single positive population. But sometimes, you will discover that a second distinct population has appeared (Figure C). It can be due to primer dimers and non-specific amplification.

Multiple fluorescence populations may prevent from setting a proper threshold.

Figure C. In this assay, a second population is observed between the negative (lower grey band) and the positive (blue).

Various methods can be employed to limit this phenomenon such as optimization of primer design, increase of annealing temperature and touchdown PCR.

This specificity issue may be fixed:

• The hybridization temperature is potentially too low so make sure you pick the highest temperature that gives you optimal separability while minimizing the rain
• Have you thought about performing a touchdown PCR?
• Is this non-specific population really an issue ? In terms of quantification, you can just decide to place the threshold above it as we have done in Figure C. The non-specific population amplified will just be considered part of the negative population. The results will thus be calculated only for the amplified partitions that show a reaction efficiency consistent with a specific hybridization of the primers and probes. Please note that this is not the optimal way of analyzing your data since it can have an impact on precision and sensitivity.
• Re-design your primers. To do so, you may use online tools to check that your primers and probes do not hybridize elsewhere in the genome.

Lievens, A., Jacchia, S., Kagkli, D., Savini, C., Querci, M. Measuring Digital PCR Quality: Performance Parameters and Their Optimization. PLoS One. 2016 May 5;11(5):e0153317. doi: 10.1371/journal.pone.0153317. eCollection 2016. PMID:  271494

## Performing Digital PCR Hands-On

Each digital PCR platform has its own hands-on protocol. In this item, we have chosen to describe protocols used on the Naica™ System and on the QX200™ Droplet Digital™ PCR System.

For both systems, we will assume that you have already prepared your PCR mix and divided it into 8 tubes: one per sample. You have also loaded your 8 samples.

### 1. Naica™ System:

Since the Sapphire chips are already prefilled with oil, you have only one last step before launching the PCR reaction.

To do so, you will need :

• 2 Sapphire Chips
• tips and pipette adapted for 25 µL
• 8 tall white caps (provided in the Sapphire Chips box)

1.  Unpack the Sapphire Chips and remove the white caps
2. Gently pipette your 25 µl of PCR mix containing your samples into each inlet port
3. Finally, seal each inlet port with the tall white caps

The hands-on part is done and you are now ready to place your Sapphire Chips into the Naica™ Geode where the partition generation and the thermal cycling will occur.

A short movie of the workflow is also available here!

### 2. QX200™ Droplet Digital™ PCR System:

Here, we describe the manual protocol. A faster way to proceed would be to use the QX200™ AutoDG™ Droplet Digital™ PCR System where the following steps are automated and integrated.

Two more steps before launching the PCR reaction :

#### A. Partitions generation

To do so, you will need :

• 1 x DG8™ Cartridges for QX200™/ QX100™ Droplet Generator
• 1x DG8™ Cartridges holder for QX200™/ QX100™ Droplet Generator
• 1 x DG8™ Gasket for QX200™/ QX100™ Droplet Generator
• 1 x QX200™ droplet generator
• 1x Droplet Generation Oil for Probes
• Rainin pipettes and tips adapted for 20µL and 70 µL

1.  Take the DG8™ Cartridges holder for QX200™/ QX100™ Droplet Generator. Open it by pressing the latches in the middle and sliding the two parts. Place one cartridge inside. Slide again in the original position to close
2. Pipette your 20µL of PCR mix containing your sample in the middle wells of the cartridge. Then, pipette 70µL of droplet generation oil in the bottom wells
3. Close the cartridge with one dedicated gasket
4. Open the QX200™ Droplet generator by pressing the button on the top. Place the cartridge holder and close the droplet generator by pressing again the button

Partitions are now generated! The second and last hands-on part starts now :

#### B. Partitions transfer

To do so, you will need:

• 1 x ddPCR 96-Well Plate
• 1 x Pierceable foil plate seal
• 1 x PCR plate sealer
• Rainin pipettes and tips adapted for 40 µL

Let’s finish!

1. Recover the DG8™ Cartridges holder from the Droplet Generator and remove the gasket. Do not open the cartridge holder.
2. Gently pipette the 40µL of generated emulsion (top wells of the cartridge) and smoothly deliver it into a ddPCR 96-Well plate.
3. Place the Pierceable foil on the ddPCR 96-well plate and proceed to the sealing part in the PCR plate sealer.
4. Finally, place your ddPCR 96-well plate in the thermal cycler and launch your ddPCR program.

### Determining the Limit of Blank (LOB):

As false positives are assay dependent and may arise from several different sources including polymerase error, DNA contamination, or non-stringent PCR or probe conditions, it is necessary to determine the LOB of the mutant. The LOB is defined according to Armbruster and Pry (2009) as “the highest apparent analyte concentration expected to be found when replicates of a blank sample containing no analyte are tested” for a given confidence level (typically 95%). The mutant LOB can be determined by performing a total of n≥30 replicate experiments containing a background of wild-type DNA and recording the number of observed “false positive events”. In the drop-off case, a false positive event is a partition that is simultaneously positive for the reference channel and negative for the wild-type channel.

Once the LOB is determined, a KRAS mutant is said to be detected if the number of observed positive events for the mutant is strictly greater than the LOB.

## 6. How to analyze the dPCR data?

Note: The sample partitions produced using the Naica™ System are referred to as “droplets” since the partitions are arranged in a droplet crystal which is typical for the Naica™ System.

#### a. Quality controls

The typical dPCR analyzing software uses several quality controls for data analysis. However, these two criteria should be carefully monitored:

• The total number of partitions analyzed: The uncertainty of the concentration measured by the software is lowered as the number of analyzed droplets increases. The number of partitions obtained for this experiment was between 21,000 and 27,000 per well.
• The ability to visualize the spatial position and the fluorescence levels of the partitions allows the users to have high confidence in the included and excluded partitions for robust results. This data transparency also allows both to verify that the target distribution in the droplets follows a Poisson distribution and to monitor the intensity and spatial uniformity of the fluorescent signal in the droplets. Stilla’s Crystal Miner software allows this type of monitoring and is fully transparent.

#### b. Setting the threshold

• Three different populations are obtained from a drop-off experiment (Figure 2). The 1st quadrant population provides the number of droplets including wild-type targets, the 4th quadrant population provides the number of droplets including mutants and the 3rd quadrant population is the number of double negative droplets. Quadrant 2 is empty by design.
• For a clear separation between the three populations of interest, it is important to verify that a threshold has been correctly set for the blue and red channels. This threshold is set automatically by the Crystal Miner software.

Figure 2. Crystal Miner 2D dot-plot generated using Crystal Miner software detects droplets containing DNA. The droplets are divided into four quadrants based on the fluorescence intensity of the probes for the red and blue color channels. The blue and the red lines are the thresholds for the blue and the red channels, respectively. The violet droplets in the 1st quadrant represent the double-positive DNA population (P11) produced by the hybridization of both the WT (Cy5) and the reference (FAM) probes. The black droplets in the 3rd quadrant indicate the double negative population (P00), containing neither WT nor mutant DNA. The deep blue droplets in the 4th quadrant represent the single positive, mutant population (P10) detected by the reference probe (FAM).

#### c. Calculating the concentration of the target DNA

• The Mutant Allelic Fraction (MAF) is the ratio of the estimated concentration of mutant DNA and the estimated concentration of the total DNA (see the formula in Results and Conclusion).
• It is important to note that co-encapsulation of the mutant and WT DNA can occur randomly in some droplets, leading to double-positive droplets that are indistinguishable from the WT DNA-only containing droplets. As such, it is important to deduct this double-positive population when calculating the mutant concentration so as not to underestimate the mutant DNA concentration.
• The concentration of the mutant DNA can be calculated from the proportion of droplets that are single positive for FAM among the droplets that are not double-positive (i.e. single FAM positive or double negative).
• The concentration of total DNA can then be obtained by summing the wild-type concentration and the mutant concentration.

## 7. Results and Conclusions

The data can be exported from the Crystal Miner software (or the software used for data acquisition) as an excel sheet. Following the MAF can be calculated.

A detailed and state-of-the-art description of the method for calculating the confidence interval of a ratio of concentrations can be found in the literature, see:

https://www.ncbi.nlm.nih.gov/pubmed/18682853

Briefly,

$$C_{mut} = -\frac{1}{v} \: \ln(1-\frac{P_{10}}{P_{10} + P_{100}})$$

where $$v$$ is the average partition volume (in µl) and $$C$$ is the mutant concentration (in copies/µl).

And,

$$C_{WT} = -\frac{1}{v} \: \ln(1-\frac{P_{11}}{P_{11} + P_{00}+P_{10}})$$

To determine the MAF, this formula can be used: $$MAF_{dropoff} = \frac{C_{Mut}}{C_{WT} + C_{Mut}}$$

For this experiment, the results are as shown in the results table below. Serial dilutions of mutant DNA ranging from 500 to 25 copies/µl in a background of 10,000 copies of WT DNA were performed to achieve mutant allele fractions ranging between 5- 0.25%. The final result was a detection level of 0.3% MAF in a background of ~330 copies/µl of the sample.

Also, for our data, the relative uncertainty was obtained from the WT and the mutant. The relative uncertainty here has a high value owing to the large uncertainty of the mutant fraction because the sample we assayed has a low concentration of rare mutations. This value points to the need for high precision and sensitivity, as the level of detection depends on the sensitivity of the digital PCR system used.

Results table

To summarize a drop-off assay:

• One of the major advantages of running a Drop-off assay is the power to detect multiple proximal genetic lesions (insertions, deletions, and nucleotide substitutions) within a short genomic interval using a single assay.
• For therapeutic monitoring in cancer patients, a set of predictive genetic markers is monitored in clinical settings. For example, in colorectal cancer patients, KRAS and NRAS proto-oncogene mutations indicate resistance to anti-EGFR.
• In this tutorial, one set of WT and Reference probes were used to detect up to 7 mutations that occur in colorectal carcinoma in the KRAS.
• A drop-off assay is a cost-effective and rapid assay for the simultaneous screening of a wide range of clinically relevant genetic alterations.

For detailed instructions and guidelines for reporting and publishing data and results obtained from a Digital PCR experiments, see the MIQE Guidelines Item here:

## MIQE Guidelines

In order to ensure experimental transparency, consistency between research laboratories and thus, maintain a high level of integrity in publications, a guideline for real-time PCR experiments has been edited in 2009 by an international research team [PMID: 19246619].

Considering the increasing number of publications in the Digital PCR field, the MIQE (for Minimum Information for Publication of Quantitative real-time PCR Experiments) guideline has been updated for digital PCR experiments under the name “the Minimum Information for the Publications of Quantitative Digital PCR Experiments guidelines (dMIQE)”[PMID: 23570709].

Having a look at this guideline before starting designing an experiment can be very informative. Indeed, as an example of what you can read in the dMIQE, you will find, below (Table 1), a checklist you might follow for performing a digital PCR experiment. Finally, all items are categorized as essential (E) or desirable (D) for manuscript submission. Thus, citing the following dMIQE in your publication will be a guarantee of quality, for reviewers and readers.

 Table 1. dMIQE checklist for authors, reviewers and editors.a Item to check Importance Item to check 2 Importance Experimental design dPCR oligonucleotides Definition of experimental and control groups. E Primer sequences and/or amplicon context sequence.b E Number within each group. E RTPrimerDB (real-time PCR primer and probe database) identification number. D Assay carried out by core lab or investigator’s lab ? D Probe sequences.b D Power analysis. D Location and identify of any modifications. E Sample Manufacturer of oligonucleotides. D Description. E Purification method. D Volume or mass of sample processed. E dPCR protocol Microdissection or microdissection. E Complete reaction conditions. E Processing procedure. E Reaction volume and amount of RNA/cDNA/DNA E If frozen-how and how quickly? E Primer, (probe), Mg ++ and dNTP concentrations. E If fixed- with what, how quickly? E Polymerase identity and concentration. E Sample storage conditions and duration (especially for formalin-fixed, paraffin-embedded samples). E Buffer/kit catalogue no. and manufacturer. E Nucleic acid extraction Exact chemical constitution of the buffer D Quantification-instrument/method. E Additives (SYBR green I, DMSO, etc.). E Storage conditions: temperature, concentration, duration, buffer. E Plates/tubes Catalogue No and manufacturer. D DNA or RNA quantification E Complete thermocycling parameters. E Quality/integrity, instrument/method, e.g. RNA integrity/R quality index and trace or 3’:5’. E Reaction setup. D Template structural information. E Gravimetric or volumetric dilutions (manual/robotic). D Template modification (digestion, sonication, preamplification, etc.) E Total PCR reaction volume prepared. D Template treatment (initial heating or chemical denaturation). E Partition number. E Inhibition dilution or spike. E Individual partition volume. E DNA contamination assessment of RNA sample. E Total volume of the partitions measured (effective reaction size). E Detail of DNase treatment where performed. E Partition volume variance/SD. D Manufacturer of reagents used and catalogue number D Comprehensive details and appropriate use of controls. E Storage of nucleic acid: temperature, concentration, duration, buffer. E Manufacturer of dPCR instrument. E RT (if necessary) dPCR validation cDNA priming method + concentration. E Optimization data for the assay. D One- or 2-step protocol. E Specificity (when measuring rare mutations, pathogen sequences etc.) E Amount of RNA used per reaction E Limit of detection of calibration control. D Detailed reaction components and conditions. E If multiplexing, comparison with singleplex assays. E RT efficiency. D Data analysis Estimated copied measured with and without addition of RT.b D Mean copies per partition (ʎ or equivalent). E Manufacturer of reagents used and catalogue number. D dPCR analysis program (source, version). E Reaction volume (for 2-step RT reaction). D Outlier identification and disposition. E Storage of cDNA : temperature, concentration, duration, buffer. D Results of no-template controls. E dPCR target information Examples of positive(s) and negative experimental results as supplemental data. E Sequence accession number. E Where appropriate, justification of number and choice of reference genes. E Amplicon length. E Number and concordance of biological replicates. D In silico specificity screen (BLAST, etc.) E Number and stage (RT or dPCR) of technical replicates. E Pseudogenes, retropseudogenes or other homologs? D Repeatability (intraassay variation). D Sequence alignment. D Reproductibility (interassay/user/lab etc. variation). D Secondary structure analysis of amplicon and GC content. D Experimental variance or Cl.d E Location of each primer by exon or intron (if applicable). E Statistical methods used for analysis. E Where appropriate, which splice variants are targeted? E Data submission using RDML (Real-time PCR Data Markup Language). D a All essential information (E) must be submitted with the manuscript. Desirable information (D) should be submitted if possible. b Disclosure of the primer and probe sequence is highly desirable and strongly encouraged. However, since not all commercial predesigned assay vendors provide this information, when it is not available assay context sequences must be submitted [Bustin et al. (48)]. c Assessing the absence of DNA using a no-RT assay (or where RT has been inactivated) is essential when first extracting RNA. Once the sample has been validated as DNA free, inclusion of a no-RT control is desirable, but no longer essential. d When single dPCR experiments are performed, the variation due to counting error alone should be calculated from the binomial (or suitable equivalent) distribution.

Congratulations on successfully performing this digital PCR experiment!

Want to discover more dPCR experiments? Check out the other tutorials!