Lossless and Contamination-Free Digital PCR

Abstract

20The realization of the vast potential of digital PCR (dPCR), to provide extremely 21 accurate and sensitive measurements in the clinical setting, has thus far been 22hindered by challenges such as assay robustness and high costs. Existing 23 popular dPCR platforms that target the clinic have not reached wide-spread 24 adoption, due to problems with sample loss and risk of contamination during 25 sample preparation, compartmentalization, and transfers; limitations of 26 dynamic range and signal-to-noise in the result readout also restricts broad 27 applications. Here we introduce a lossless and contamination-free dPCR 28 technology termed CLEAR-dPCR, which addresses these challenges by 29completing the dPCR sample preparation, PCR reaction, and readout all in one 30tube. We achieve this by adjusting the refractive index of the aqueous PCR mix 31to make the emulsion optically transparent, and devised a light-sheet 32 microscope to capture 3D images of the cleared emulsion for results readout. 33This approach demonstrates improved accuracy over existing dPCR platforms, 34and enables a greatly increased dynamic range to be comparable to that of 35 real-time quantitative PCR (qPCR). CLEAR-dPCR is an easy to operate, 36 sensitive and accurate dPCR platform that we envision will fulfill the potential 37 of dPCR for routine use clinical diagnosis. 38 39 2 Main Text 40 41The desire for better quantitative assessment of genetic information is always 42increasing. Techniques for sequence-specific nucleic acid quantification are 43 key tools in biology and medicine, especially in scenarios involving rare genetic 44 variant detection where both high sensitivity and high specificity are warranted 45 1-3

Centrifugal Microfluidic Integration of 4-Plex ddPCR Demonstrated by the Quantification of Cancer-Associated Point Mutations

“…In the recent past, centrifugal microfluidics developed into a mature technology which enabled the efficient miniaturization, parallelization and integration of assays for both sample preparation and analysis [13][14][15]. In the past, several groups have presented centrifugal, microfluidic cartridges that were able to emulsify a reaction mix [6,9,[16][17][18][19][20]. We present a centrifugal, microfluidic, single-use cartridge (LabDisk) with 12 identical ddPCR units.…”