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Real Time RT-PCR

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The real-time quantification of the number of amplified products is...

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Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Published on: September 24, 2015

Fluorescence-coded logarithmic-dilution digital droplet PCR for ultrawide-dynamic-range nucleic acid quantification.

Qingyuan Shi¹, Jie Li², Chunchen Liu³

¹State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai, 200050, China; School of Physical Science and Technology, ShanghaiTech University, Shanghai, 201210, China.

Biosensors & Bioelectronics

|September 26, 2023

Summary

Fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) significantly expands the dynamic range for nucleic acid detection. This advancement overcomes limitations of current digital PCR (dPCR) methods, enhancing clinical applicability.

Keywords:

Biosensing Digital PCR Droplet Dynamic range HPV Nucleic acid

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Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Published on: June 26, 2016

Area of Science:

Molecular Biology
Biotechnology
Analytical Chemistry

Background:

Digital PCR (dPCR) offers absolute quantification and high sensitivity for nucleic acid detection.
Conventional dPCR has a limited dynamic range (4-5 log) compared to quantitative PCR (qPCR), restricting its clinical use.
Expanding the dynamic range of dPCR is crucial for broader applications.

Purpose of the Study:

To develop a novel digital PCR method with an expanded dynamic range.
To overcome the dynamic range limitations of conventional dPCR for improved clinical applicability.
To present fluorescence-coded logarithmic-dilution digital droplet PCR (Flodd-PCR) as a solution.

Main Methods:

Utilized a microfluidic chip for multi-step serial dilution (20-925 folds) and droplet generation.
Divided approximately 20,000 droplets into 4 groups, each with a unique DNA template dilution factor.
Employed fluorescence-coded droplets for in silico clustering and independent analysis based on dilution indicators.

Main Results:

Achieved a 7-order magnitude dynamic range, exceeding conventional dPCR by over 2 orders.
Demonstrated detection of 4-20,000,000 copies/μL of synthetic human papillomavirus (HPV) DNA.
Flodd-PCR outperformed standard dPCR in analyzing clinical HPV samples.

Conclusions:

Flodd-PCR significantly enhances the dynamic range of digital PCR, approaching that of qPCR.
The method is compatible with existing dPCR systems, ensuring broad practicality.
Flodd-PCR shows promise for various applications where conventional dPCR is currently used, particularly in clinical diagnostics.