"A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets" 

Reya Ganguly, under Prof. Chang-Soo Lee, has introduced an innovative approach to nucleic acid quantification in microdroplets, presenting a significant advancement in digital droplet PCR (dPCR) technology. Published in ACS Applied Bio Materials on April 24th, 2024, their work presents a method that circumvents the limitations posed by Poisson statistics in ddPCR, offering a Poisson-independent means of quantification. 

In their paper titled "A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets", researchers introduce an advanced microdroplet-based competitive PCR platform, offering an efficient method for quantifying DNA within microfluidic devices, independent of Poisson statistics. Despite inherent challenges in droplet-based systems, the study shifts focus from binary-end point quantification to leveraging fluorescent intensity for precise DNA quantification.

Their methodology aims to determine the exact number of molecules within each droplet and estimate average total fluorescent intensity to establish an equivalence point. This crucial point occurs when concentrations of competitor and target DNA are equal, resulting in a C/T ratio of 1, with competitor DNA concentration acting as a known factor.

In this approach, target and competitor DNA compete for primer binding during amplification, with the resulting competitor DNA products serving as the measured indicator. The concentration of target DNA is subsequently determined by the competitor DNA concentration that yields an equivalent molar amount of target DNA product, termed the Equivalence Point (E.P.), where C/T equals 1.

To illustrate, a microfluidic device was designed to establish a concentration gradient of competitor DNA alongside a consistent concentration of target DNA. Post-amplification and imaging analysis of collected droplets allowed the researchers to pinpoint the Equivalence Point (E.P.).

The findings indicate that as the concentration of target DNA decreases, the slope of the fluorescence intensity versus target DNA concentration graph steepens. However, once the target DNA concentration surpasses that of the competitor DNA (Cmax), the intensity fails to exceed the Equivalence Point, plateauing instead. This phenomenon stems from the inverse relationship between fluorescence intensity and target DNA concentration.

The platform offers valuable insights into sample quality and quantity, particularly when combined with Equivalence Point analysis and scrutiny of slope variations concerning target DNA concentration. Importantly, the quantification method, which averages intensity across droplets, differs from ddPCR, where quantification is binary, categorizing each droplet as positive or negative. 

This feature addresses challenges associated with misclassification, presenting a dependable alternative for nucleic acid quantification.

This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (Nos. 2021R1A2C3004936 and 2021R1A5A8032895).

Related papers: A Poisson-Independent Approach to Precision Nucleic Acid Quantification in Microdroplets

□ Link: https://doi.org/10.1021/acsabm.4c00350