This work was supported by Institut Curie SiRIC (grant INCa- DGOS-4654).&#160;The authors wish also to thank Caroline Hego for her contribution to the video.

<ol>
	<li>Alexandrov, L. B., Nik-Zainal, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signatures+of+mutational+processes+in+human+cancer.">Signatures of mutational processes in human cancer.</a> Nature. 500 (7463), 415-421 (2013).</li><li>Amado, R. G., Wolf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-Type+KRAS+Is+Required+for+Panitumumab+Efficacy+in+Patients+With+Metastatic+Colorectal+Cancer.">Wild-Type KRAS Is Required for Panitumumab Efficacy in Patients With Metastatic Colorectal Cancer.</a> Journal of Clinical Oncology. 26 (10), 1626-1634 (2008).</li><li>Karapetis, C. S., Khambata-Ford, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K-ras+mutations+and+benefit+from+cetuximab+in+advanced+colorectal+cancer.">K-ras mutations and benefit from cetuximab in advanced colorectal cancer.</a> New England Journal of Medicine. 359 (17), 1757-1765 (2008).</li><li>Postel, M., Roosen, A., Laurent-Puig, P., Taly, V., Wang-Renault, S. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet-based+digital+PCR+and+next+generation+sequencing+for+monitoring+circulating+tumor+DNA:+a+cancer+diagnostic+perspective.">Droplet-based digital PCR and next generation sequencing for monitoring circulating tumor DNA: a cancer diagnostic perspective.</a> Expert Review of Molecular Diagnostics. 18 (1), 7-17 (2018).</li><li>Saliou, A., Bidard, F. -. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+for+triple-negative+breast+cancer+diagnosis+and+treatment+decisions.">Circulating tumor DNA for triple-negative breast cancer diagnosis and treatment decisions.</a> Expert Review of Molecular Diagnostics. 16 (1), 39-50 (2015).</li><li>Decraene, C., Silveira, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Hotspot+Mutations+Scanning+by+Single+Droplet+Digital+PCR.">Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.</a> Clinical chemistry. 64 (2), 317-328 (2018).</li><li>Untergasser, A., Cutcutache, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primer3--new+capabilities+and+interfaces.">Primer3--new capabilities and interfaces.</a> Nucleic Acids Research. 40 (15), e115 (2012).</li><li>Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M., Shendure, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+Comprises+an+In+Vivo+Nucleosome+Footprint+that+Informs+Its+Tissues-Of-Origin.">Cell-free DNA Comprises an In Vivo Nucleosome Footprint that Informs Its Tissues-Of-Origin.</a> Cell. 164 (1-2), 57-68 (2016).</li><li>Bidshahri, R., Attali, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Detection+and+Resolution+of+BRAF+V600+Status+in+Colorectal+Cancer+Using+Droplet+Digital+PCR+and+a+Novel+Wild-Type+Negative+Assay.">Quantitative Detection and Resolution of BRAF V600 Status in Colorectal Cancer Using Droplet Digital PCR and a Novel Wild-Type Negative Assay.</a> The Journal of Molecular Diagnostics. 18 (2), 190-204 (2016).</li><li>Attali, D., Bidshahri, R., Haynes, C., Bryan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ddpcr:+an+R+package+and+web+application+for+analysis+of+droplet+digital+PCR+data.">ddpcr: an R package and web application for analysis of droplet digital PCR data.</a> F1000Research. 5, (2016).</li><li>Forbes, S. A., Beare, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=COSMIC:+somatic+cancer+genetics+at+high-resolution.">COSMIC: somatic cancer genetics at high-resolution.</a> Nucleic Acids Research. 45 (D1), D777-D783 (2017).</li></ol>

Several authors are named inventors of two patent applications (EP17305920.5, EP18305277.8) related to ctDNA detection.

To design an efficient drop-off ddPCR assay, optimization is crucial, and the protocol must be followed carefully. Each combination of primers and probes has a unique PCR reaction efficiency. Thus, an individual assay has to be carefully validated on control samples before being used on valuable test samples. Optimization and validation are important to certify peak signal detection and assess specificity and sensitivity. As described in the protocol, all mutations located in a target region covered by the &#34;probe 2&#...

Thousands of somatic mutations related to cancer development have been reported1. Among these, a few are predictive markers of the efficacy of targeted therapy 2,3 and genetic screening of these mutations is now routine clinical practice. Droplet digital PCR (ddPCR) technology can be used to monitor for the presence or the absence of mutations with high detection accuracy and is highly compatible with non-invasive liquid biopsies4,5. However, current ddPCR assays are primarily designed to detect mutations known a priori. This severely limits the use of ddPCR as a discovery tool when the mutation is unknown. Indeed, in the conventional ddPCR design, a hydrolysis probe recognizing the wild-type allele (WT Probe) competes with a specific probe recognizing the mutant allele (MUT Probe) (Figure 1A). The probe with higher affinity hybridizes to the template and releases its fluorophore, indicating the nature of the corresponding allele. The fluorescence data obtained for each droplet can be visualized in a scatter plot representing the fluorescence emitted by the WT and MUT probes in different dimensions. A schematic representation of a typical result for a conventional ddPCR assay is presented in Figure 1A. In this example, the blue cloud corresponds to the droplets containing WT alleles identified by the WT probe, whereas the red cloud corresponds to droplets in which the MUT allele has been amplified and identified by the MUT probe. Depending on the quantity of DNA loaded in the reaction, double positive droplets containing both WT and MUT amplicons can appear (green cloud). The light grey cloud corresponds to empty droplets.
Since most platforms allow for the detection of a limited number of fluorophores (usually 2, but up to 5), throughput of conventional ddPCR is limited. Therefore, targeting regions with multiple adjacent mutations requires design of technically challenging multiplex ddPCR assays or the use of multiple reactions targeting each mutation in parallel. The drop-off ddPCR strategy, overcomes this limitation as it can potentially detect any genetic alteration within a target region using a unique pair of WT hydrolysis probes. The first probe (Probe 1) covers a non-variable sequence, adjacent to the target region and the second probe (Probe 2) is complementary to the WT sequence of the target region where mutations are expected (Figure 1B). While the first probe quantifies the total amount of amplifiable molecules in the reaction, the second probe discriminates WT and MUT alleles by sub-optimal hybridization to mutant sequences. Probe 2 can identify multiple types of mutations in the target region (single or multiple nucleotide substitutions, deletion, etc.)6. As in conventional ddPCR, the light grey cloud corresponds to droplets containing no DNA molecules. It is important to recall that in this type of assay, optimal separation of WT and MUT clouds is dependent on the quantity of DNA loaded in the reaction, since droplets containing both WT and MUT target alleles cannot be distinguished from droplets containing only the WT form. Therefore, this assay requires that most droplets contain no more than one copy of the targeted gene.

<table><tbody><tr><td>K2EDTA tube (color code : lavender)</td><td>BD</td><td>367863</td><td>EDTA tubes are used to obtain a whole blood or EDTA plasma sample</td></tr><tr><td>QIAamp Circulating Nucleic Acid Kit (manual protocol)</td><td>Qiagen</td><td>55114</td><td>For isolation of free-circulating DNA from human plasma</td></tr><tr><td>QIAsymphony DSP Circulating DNA Kit (automated protocol)</td><td>Qiagen</td><td>937556</td><td>For isolation of free-circulating DNA from human plasma</td></tr><tr><td>QIAsymphony SP system</td><td>Qiagen</td><td>9001297</td><td>fully integrated and automated system for cfDNA, DNA or RNA purification</td></tr><tr><td>QIAvac 24 Plus</td><td>Qiagen</td><td>19413</td><td>For purification of up to 24 cfDNAs simultaneously</td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td>Q33226</td><td>The Qubit fluorometer is a benchtop fluorometer for the quantitation of DNA</td></tr><tr><td>QX100 or QX200 reader</td><td>Bio-Rad</td><td>186-3003 or186-4003, respectively</td><td>The reader measures fluorescence intensity of each droplet and detects the size and shape as droplets pass the detector</td></tr><tr><td>QX100 or QX200 droplet generator</td><td>Bio-Rad</td><td>186-3002 or 186-4002, respectively</td><td>Instrument used for droplet generation</td></tr><tr><td>C1000 thermal cycler</td><td>Bio-Rad</td><td>1851196</td><td>Modular thermal cycler platform, includes C1000 Touch thermal cycler chassis, 96-well fast reaction module</td></tr><tr><td>Plate sealer</td><td>Bio-Rad</td><td>181-4000</td><td>PX1&amp;trade; PCR plate sealer</td></tr><tr><td>DG8 cartridge holder</td><td>Bio-Rad</td><td>186-3051</td><td>Positions and holds the DG8 cartridge in the instrument for droplet generation</td></tr><tr><td>Droplet generator cartridges and gaskets</td><td>Bio-Rad</td><td>186-4007</td><td>Microfluidic DG8 cartridge used to mix sample and oil to generate droplets; DG8 gaskets seal the cartridge to prevent evaporation and apply the pressure required for droplet formation</td></tr><tr><td>PCR supermix</td><td>Bio-Rad</td><td>186-3010</td><td>ddPCR supermix for probes (no dUTP)</td></tr><tr><td>96-well PCR plates</td><td>Eppendorf</td><td>951020362</td><td>96-well semi-skirted plates</td></tr><tr><td>Foil seal</td><td>Bio-Rad</td><td>181-4040</td><td>Pierceable foil heat seal</td></tr><tr><td>ddPCR Droplet Reader Oil</td><td>Bio-Rad</td><td>186-3004</td><td>oil used in the read</td></tr><tr><td>Droplet Generation Oil for Probes</td><td>Bio-Rad</td><td>186-3005</td><td>oil for droplet generation</td></tr><tr><td>DNA loBind Tube 1.5 mL</td><td>Eppendorf</td><td>0030 108.051</td><td>Eppendorf LoBind Tubes maximize sample recovery by significantly reducing sample-to-surface binding</td></tr><tr><td>Multiplex I cfDNA Reference Standard Set</td><td>HORIZON</td><td>HD780</td><td>The Multiplex I cfDNA Reference Standards are highly-characterized, biologically-relevant reference materials used to assess the performance of cfDNA assays that detect somatic mutations</td></tr><tr><td>QUBIT DSDNA HS ASSAY KIT, 500</td><td>Life Technologies</td><td>Q32854</td><td>The HS assay is highly selective for double-stranded DNA (dsDNA) over RNA and is designed to be accurate for initial sample concentrations from 10 pg/&amp;micro;L to 100 ng/&amp;micro;L</td></tr><tr><td>Qubit Assay Tubes</td><td>Life Technologies</td><td>Q32856</td><td>Qubit assay tubes are 500 &amp;micro;L thin-walled polypropylene tubes for use with the Qubit Fluorometer</td></tr></tbody></table>

single droplet digital polymerase chain reaction for comprehensive and simultaneous detection of mutations in hotspot regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

In a proof-of-concept study, KRAS exon 2 mutations (codons 12 and 13) and EGFR exon 19 deletions were investigated in FFPE tissues and plasma samples from cancer patients using the drop-off ddPCR strategy6.
The KRAS drop-off probe interrogated a 16 bp region encompassing multiple mutations in exon 2 of the KRAS gene, which harbor more than 95% of the known KRAS </em...

Watch this Scientific Journal Video about Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions at JoVE.com

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

single-droplet-digital-polymerase-chain-reaction-for-comprehensive

Research

Education

JoVE Journal

JoVE Core

Molecular Biology

Genetics

Unit 1

DNA, Cells, and Evolution

SCIENTISTS IN ACTION

13.0K Views.  Institut Curie, PSL Research University. Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Video: Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Cancer Research

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Optimizing Low-Frequency Mutation Detection with Wild-Type Blocking PCR

Wild-type Blocking PCR Combined with Sanger Sequencing for Detection of Low-frequency Somatic Mutation

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting for drug resistance mutations and circulating tumor cell mutations during therapy. Traditional Sanger sequencing has 5%-20% wild-type mutation detection, so its limit of detection cannot meet the corresponding requirements. The wild-type blocking technologies that have been reported to overcome this include blocker displacement amplification (BDA), non-extendable locked nucleic acid (LNA), hot-spot-specific probes (HSSP), etc. These technologies use specific oligonucleotide sequences to block wild-type or recognize wild-type and then combine this with other methods to prevent wild-type amplification and amplify mutant amplification, leading to characteristics like high sensitivity, flexibility, and convenience. This protocol uses BDA, a wild-type blocking PCR combined with Sanger sequencing, to optimize the detection of RHOA G17V low-frequency somatic mutations, and the detection sensitivity can reach 0.5%, which can provide a basis for MRD monitoring of angioimmunoblastic T-cell lymphoma.

Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues

This article introduces the application of a low-frequency detection method based on Sanger sequencing in angioimmunoblastic lymphoma. Provide a basis for applying this method to other diseases.

When monitoring minimal residual disease (MRD) after tumor treatment, there are higher requirements of the lower limit of detection than when detecting ...

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Immunology and Infection

Duplex Digital PCR for Simultaneous Quantification of Dual Genetic Markers

Source: Cao, Y., et al. <a href="https://app.jove.com/v/53611/">A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters.</a> J. Vis. Exp. (2016)
In this video, we demonstrate the duplex digital PCR (ddPCR) technique &#8212; a modification of the traditional PCR technique that is useful in detecting two different genetic markers simultaneously. A single PCR reaction is partitioned into nanoliter-sized emulsified droplets that are independently amplified, and the detection of differently colored fluorescence amplification signals from the fraction of droplets is used to compute the initial concentration of the target sequences.

Source: Cao, Y., et al. A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in ...

JoVE Encyclopedia of Experiments

Biological Techniques

PCR Techniques

High-Resolution Melting PCR to Determine Sequence Variations in Mutant DNA Sequences

Source: Kisserli, A., et al. <a href="https://app.jove.com/v/56012">High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s Disease Gene Susceptibility Assessment.</a> J. Vis. Exp. (2017).
In this video, we describe a high-resolution melting PCR technique to determine the length polymorphism of DNA fragments of varying sequence lengths, based on melting temperature analysis.

Source: Kisserli, A., et al. High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#39;s ...

Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Source: Kahyo, T., et al. <a href="https://app.jove.com/v/58199">Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format.</a> J. Vis. Exp. (2018).
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.

Source: Kahyo, T., et al. Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the ...

Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

Before beginning the droplet digital PCR procedure, clean the bench space and equipment with 10% bleach and 70% ethanol and gently vortex and briefly centrifuge all of the reagents except the droplet-stabilizing oil. Confirm that the compressed nitrogen gas cylinder is attached to the droplet-generating instrument and that the cylinder tank is set at 90 pounds per square inch. Add 38 microliters of digital PCR reaction mixture directly into the bottom of each well of an 8-well PCR tube strip removing any bubbles with a clean pipette tip.
Add 12 microliters of pre-amplified DNA sample in triplicate to the appropriate wells of the tube strip. Gently pipette the full volume 10 times to mix the reaction mixture with the DNA sample and pipette the full volume from each well into the corresponding A through H channels of a droplet-generating instrument chip. Insert a new PCR tube strip into the droplet-generating instrument and scan the droplet-generating instrument chip ID into the software on the instrument computer.
Then, click &#34;Start the run&#34; to begin dropletizing samples. When the dropletization is complete, remove the PCR tube strip and apply tube strip caps. Transfer the tube strip to a thermal cycler and balance the tube with another tube strip containing 80 microliters of water per well. Then, run the thermal cycler under the appropriate thermal cycling conditions. Replace the strip caps with high-speed caps.
When the thermal cycling is complete, transfer the tube strip to the quantification instrument. Click &#34;Setup a run&#34; on the instrument software and place the tube strip into the quantification instrument. Place the metal shield on top of a new quantification instrument chip. Scan the chip ID into the instrument and insert the chip into the machine. Close the lid of the instrument. In the computer software, enter a name for the digital PCR run and for each of the eight channels. Then, select &#34;Fast mode&#34; and click &#34;Start&#34; to begin the quantification.

Cancers of the Nervous System

In vitro studies

EoE Sub Articles

Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

To begin with, prepare 25 microliters of the digital droplet PCR or ddPCR sample mix by adding ddPCR super mix, forward and reverse primers, HEX or FAM probes, DNA, and water in the proportions described in the text. Then, thoroughly mix the reaction by vortexing.
Using a 50-microliter multichannel pipette, load 20 microliters of the ddPCR sample mix into the middle row of the cartridge. Then, use a 200-microliter multichannel pipette to load 70 microliters of the oil into the bottom row without generating bubbles throughout the pipetting.
Place the gasket, touching only the edges, avoiding the center concaved area, and then, place the plate securely in the droplet generator, and close the cover to start the run.
To transfer the immersion mix from the top row of the cartridge into the 96-well plate, use a multichannel pipette and draw 40 microliters of liquid sample for 3 to 5 seconds at an angle of 30 to 45 degrees. Then, expel the mixture into the well slowly for over 3 seconds at a 45-degree angle, allowing it to drop down from the side, and then, go to the second stop of the pipette to expel the liquid completely. Using foil heat seals, seal the plates for 5 seconds at 180 degrees Celsius.
Place the sealed plate into the thermocycler and set the PCR conditions for NHEJ Drop-Off guidelines by setting initial denaturation at 95 degrees Celsius for 10 minutes. Then, set 40 cycles of 94 degrees Celsius for 30 seconds to denature, 55 degrees Celsius for 1 minute to anneal, and 60 degrees Celsius for 2 minutes to extend.
After 40 cycles, set hold at 98 degrees Celsius for 10 minutes, and final hold at 4 degrees Celsius. Use a ramp rate of 2 degrees Celsius per second for all steps. The annealing temperature will vary according to the probes or primer used. Place the plate securely in the droplet reader with the A1 labeled well at the top left.
Open the program and set up the plate by designating FAM as the known reference channel and HEX as the unknown one for each well. Then, change the name for each sample. Run the droplet reading experiment as direct quantification while saving the template.