Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format
August 20th, 2018
Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.
This method can help answer key questions in the genetic testing field, such as detection of mosaicism, and liquid biopsy-based genetic testing. The main advantage of this technique is high-throughput detection of low-variant allele fraction. The implications of this technique extend towards diagnosis of tumors, because detecting oncogenic mutations with high sensitivity can contribute to precision medicine.
Start this experiment by adding 10 L of DNA sample buffer to the tubes of an 8-tube strip. Then add 1 microliter of DNA ladder to the first tube of the 8-tube strip, and one microliter of sample genomic DNA to each of the remaining tubes. Use microplate attachment to mix contents of the 8-tube strip by vortexing for one minute.
Place the strip, pipet tips, and a gel device into an electrophoresis instrument. Press start button to start the run. After the complete run, check the electropherogram on the display to confirm that the lower marker contained in the DNA sample buffer is correctly assigned on the electropherogram.
If not, manually assign the marker in the electropherogram mode of the software. Then, in the region mode of the software, specify the size region of the genomic DNA to automatically calculate the concentration of DNA. Add previously-designed primers, probes, and genomic DNA to a new 8-tube strip to achieve a total volume of 15 L.Pipette up and down to mix.
Add the loading platform onto the chips built in the fresh 8-tube strip, and then place the tube strip in the autoloader. Make sure that there is a contact between the chips and the loading platform. Next, place a loading slider in the platform, and use a stopper to hold the slider off of the loader.
Pipette 15 L of the PCR mixture near the tip of the slider, and then press the loader button to run the loader for one minute. Remove the tube strip from the loader after the run, and place it in sealing enhancer. Carefully push the slide lid and the edge of the top lid.
Run the sealing enhancer for approximately two minutes. If sealing is incomplete, indicated by a puddle of liquid, repeat the run for an additional minute. Add 230 L of sealing fluid to the tubes.
Place the tube strip in the thermal cycler, and run the PCR as described in the protocol. If there is an uneven distribution of positive partitions, adjust the temperature or the duration of the PCR. To detect and analyze fluorescence intensity of the PCR products, place the tube strip on the detection jig, and add 6 mL of distilled water.
Remove any visible air bubbles using a pipette tip. Load the jig into the detector. In the detection software, select Fluorescence, Experiment, and then Sample NTC tabs, and click the RUN button to start the run.
After the complete run, confirm the position plot, histogram, and 2-D scatter plot. To collect the PCR product, remove the sealing fluid from the tube. Add 100 L of TE buffer, and vortex vigorously for 30 seconds.
Briefly centrifuge the tubes in a tabletop centrifuge, and proceed as described in the text protocol to finish collecting the PCR product. Position plots created by digital PCR show yellow HEX fluorescence in both patients and father samples, indicating presence of APC allele variant T, but not in no template control. Only patients genomic DNA contains C-variant, as shown by a green fluorescence of FAM.
As confirmed further by scatter plots, both patient and father are positive for HEX, or variant T, whereas only the patient shows the presence of variant C.Patients variant allele fraction, or VAF, calculated by DPCR, was 13.2%similar to 12.7%achieved by next generation sequencing. On the other hand, the VAFs of the patients'parents and healthy donors were less than 0.1%Electropheresis of collected and concentrated DPCR products confirm the size of the predicted fragment to be 123 base pairs in patients'and parents'DNA. While attempting this procedure, it is important to remember to keep the bench clean by, for example, using a fan filter unit.
Overview
This article discusses the application of digital polymerase chain reaction (PCR) for the sensitive detection of single nucleotide variants and DNA copy number variants. It highlights the methodology and implications of using digital PCR in genetic testing, particularly for tumor diagnosis.
Key Study Components
Area of Science
- Genetics
- Oncology
- Diagnostic Techniques
Background
- Digital PCR allows for high-throughput detection of low-variant allele fractions.
- This technique is beneficial for detecting mosaicism and in liquid biopsy-based genetic testing.
- It contributes to precision medicine by enabling the detection of oncogenic mutations with high sensitivity.
Purpose of Study
- To demonstrate the effectiveness of digital PCR in measuring rare genetic variants.
- To provide a detailed protocol for implementing this technique.
- To explore the implications of digital PCR in clinical diagnostics.
Methods Used
- Preparation of DNA samples and mixing with primers and probes.
- Electrophoresis for initial DNA analysis.
- Utilization of a thermal cycler for PCR amplification.
- Detection of PCR products using fluorescence intensity analysis.
Main Results
- Successful detection of APC allele variants in patient samples.
- Variant allele fraction (VAF) calculated by digital PCR was comparable to next-generation sequencing results.
- Electropheresis confirmed the size of PCR products.
- Demonstrated the importance of maintaining a clean workspace during the procedure.
Conclusions
- Digital PCR is a powerful tool for detecting rare genetic variants.
- This method has significant implications for cancer diagnosis and precision medicine.
- Proper execution of the protocol is crucial for accurate results.
Frequently Asked Questions
Executive Industry Relevance
Digital PCR enables precise quantification of low-variant allele fractions, supporting early detection of somatic mosaicism in cancer predisposition genes. This capability enhances target validation by providing orthogonal confirmation of rare variants identified through sequencing. The chip-in-a-tube format improves throughput for preclinical screening applications requiring high sensitivity.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by detecting low-frequency APC variants in sporadic familial adenomatous polyposis.
- Operational Value: Supports biological de-risking through orthogonal validation of mosaicism detected in patient samples.
- Predictive Value: Variant allele fraction quantification aids portfolio triage by confirming variant presence in disease-relevant systems.
Screening & Assay Development
- Assay Readiness: Chip-in-a-tube format allows eight simultaneous dPCR reactions, improving standardization for mutation screening workflows.
- Quantitative Output: Precise VAF measurement (13.2% in patient) enables reliable compound effect evaluation in preclinical models.
- Scalability: Sealing and collection procedures support reproducible partition analysis for downstream confirmation.
Translational & Preclinical Research
- Disease Relevance: Detection of APC somatic mosaicism aligns with tumorigenic pathways in colorectal cancer models.
- Translational Continuity: VAF concordance with NGS (12.7%) supports risk-adjusted advancement decisions in preclinical validation.
- Mechanistic De-risking: Fluorescence-based scatter plots confirm variant specificity, reducing false-positive risk in target selection.
Pipeline & Workflow Integration
This method fits within the discovery continuum from target validation through lead identification, providing quantitative genetic analytics for mechanistic de-risking.
- Discovery Biology: Supports hypothesis testing by quantifying APC variant allele fractions in familial cancer samples.
- Screening: Chip-in-a-tube format enables assay reproducibility and quantitative outputs for mutation screening campaigns.
- Analytics: Fluorescence intensity analysis and scatter plots deliver statistical outputs for comparing variant conditions across samples.
- Translational Research: VAF measurements connect discovery findings to preclinical continuity in adenomatous polyposis models.
- Enterprise Reuse: Standardized sealing and collection procedures allow platform reuse across multiple genetic targets.
Operational & Enterprise Impact
- Scientific Value: Predictive confidence in target validation through sensitive detection of low-variant allele fractions.
- Operational Value: Standardization via chip-in-a-tube format and sealing protocols ensures reproducibility across runs.
- Strategic Value: Reduced late-stage biological risk by confirming mosaicism early in discovery.
- Portfolio Impact: Risk-adjusted prioritization enabled by orthogonal VAF measurements matching NGS benchmarks.
Implementation Considerations
- Requires expertise in dPCR setup, fluorescence detection, and partition analysis software.
- Needs thermal cycler, detection jig, and sealing enhancer instrumentation for chip-based reactions.
- Demands cross-team standardization of sealing timing and slide lid placement to prevent partition unevenness.
- Adaptation considerations include optimizing PCR duration for even partition distribution across sample types.
- Practical limitation: Careful chip filling and sealing required to avoid over- or under-estimation of positive partitions.
