Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes.

ABSTRACT: The miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 μg/μL bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether₂ surfactant in the oil phase of 50 μm diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are th

SUBMITTER: Loveday EK 

PROVIDER: S-EPMC10016143 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature