Real-time droplet DNA amplification with a new tablet platform.
We present a novel droplet-based tablet platform for temporal polymerase chain reaction (PCR) in microliter droplets. The simple design of the device does not require extensive processing or external equipment, which allows for greater ease of use and integration as a point-of-care diagnostic. We demonstrate its functionality to perform both PCR and reverse-transcription PCR for λ phage DNA and H3 influenza RNA with ramp rates and cycle times consistent with traditional PCR thermal cyclers. We additionally investigate the effect of performing PCR in small volumes on the reaction performance by specifically examining adsorption of reagents at the oil/water interface. We determined that adsorption of Taq polymerase at the biphasic interface reduces yield and impairs reaction performance at standard concentrations. Thus, microdroplet PCR reactions require additional polymerase to achieve sufficient amplification and we project for applications utilizing nanodroplets or picodroplets like digital applications, even greater concentrations of polymerase are required to achieve desired results. Following the adsorption investigation, we evaluated the sensitivity of λ phage PCR on our platform to be less than 2.0 copies/μL with an efficiency of 104.4% and similar sensitivity for reverse-transcription PCR for influenza H3 RNA.
Center for Biomedical Engineering, School of Engineering and Division of Biology and Medicine, Brown University, Providence, Rhode Island, United States.
Stephanie L
SL
Angione
Anuj
A
Chauhan
Anubhav
A
Tripathi
D010582:Bacteriophage lambda / Q000235:genetics; D004247:DNA / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004867:Equipment Design; D009975:Orthomyxoviridae / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D012367:RNA, Viral; D004247:DNA