Droplet Digital PCR (ddPCR) Technology Platforms for Nucleic Acid Drugs

CD Formulation, a company with extensive experience in detecting nucleic acid preparations, offers digital droplet polymerase chain reaction (ddPCR) technology to accurately and quantitatively identify point mutations in samples, particularly in complex, rare, and valuable specimens.

About Droplet Digital PCR (ddPCR) Technology Platform

Droplet Digital PCR (ddPCR) is a digital PCR technology that employs a water-oil emulsion droplet system. This method operates on the principle that a PCR sample is partitioned into approximately 20,000 water-in-oil droplets using a microtitration process facilitated by a microtitre generator. In this system, nucleic acid molecules are randomly distributed among a vast number of individual droplets, with each droplet containing either one or no nucleic acid molecules to be detected.

After PCR, each droplet is analyzed to determine the proportion of PCR-positive droplets in the original sample. This data is then evaluated using Poisson statistics to ascertain the concentration of the target DNA template in the original sample.

Fig.1 Sample processing and droplet digital PCR workflow. (Zhang W, et al., 2023)

Procedure of ddPCR Technology

PCR Reaction System Construction

A PCR reaction system is prepared by mixing extracted nucleic acids with primers, probes, dNTPs, a buffer, and DNA polymerase.

Digital Titer Generation

The reaction mixture is dispensed into a microtiter generator, which creates thousands of tiny reaction cavities using a water-in-oil emulsion. Each of these microdroplets may contain either zero or one target molecule.

PCR Amplification

PCR amplification of microdroplets in a thermal cycler typically involves three steps: denaturation, annealing, and extension.

Fluorescence Signal Detection

A ddPCR instrument is utilized to detect the fluorescence signal in each microdroplet. By analyzing the fluorescence intensity, it can be determined whether each microdroplet contains the target nucleic acid, thereby facilitating digital quantitative analysis.

Data Analysis

The fluorescence signals in the droplets are subjected to statistical analysis. The actual concentration of target nucleic acid molecules is quantified by counting the number of positive (fluorescent) and negative (non-fluorescent) droplets, using the Poisson distribution model as a reference.

Requirements for ddPCR Primers and Probes

Specificity Requirements

Primers and probes must be designed with a high degree of specificity to ensure that only the target sequence is amplified and detected, thereby minimizing errors and interference.

Sensitivity Requirements

The selection of primers and probes significantly influences the sensitivity of the experiment. High-quality primers and probes can detect very low concentrations of the target sequence, enabling the experiment to reliably identify rare mutations or low-abundance targets.

Efficiency and Stability Requirements

Efficient primers and probes enhance the success rate and yield of PCR reactions, while stable primers and probes guarantee reproducible and reliable results.

Highlights of Our Droplet Digital PCR Technology Platform

• Unrivaled Accuracy - ddPCR offers extensive sample partitioning, enabling the reliable measurement of small fold differences in the number of copies of target DNA sequences within a sample.
• Eliminates PCR Bias - Reduces error rates by removing the dependence of qPCR on amplification efficiency, allowing for the detection of small (1.2-fold) differences.
• Reduced Consumable Costs - Reaction volumes in the picoliter to nanoliter range minimize reagent usage and decrease the sample volume required for each data point.
• Lower Equipment Costs - The emulsion-based reaction system allows PCR reactions to be conducted in a standard thermal cycler, eliminating the need for complex chips or microfluidics.
• Superior Partitioning - ddPCR technology produces 20,000 droplets per 20 μL sample and nearly 2 million partitioned PCR reactions in a 96-well plate. In contrast, chip-based digital PCR systems can generate only hundreds or thousands of partitions. The greater the number of partitions, the higher the accuracy.

Our Analytical Characterization Services

CD Formulation's ddPCR technology platforms have significant applications in the bioanalysis of nucleic acid drugs. Our platforms offer absolute quantitative measurements that minimize errors associated with standard curves and replication dependencies.

Publication Data

CD Formulation's ddPCR technology platform offers a highly sensitive and quantitatively accurate method for analyzing low-abundance nucleic acid molecules. Contact us to customize a professional solution tailored to your project.

How It Works

STEP 1

Submit a service request describing your specific research or development need.

STEP 2

We'll email you to provide your quote and confirm order details if applicable.

STEP 3

Execute the project with real-time communication, and deliver the final report promptly.