tetano
Editor, Senior Moderator
Anal Chem. 2012 Feb 6. [Epub ahead of print]
Real Time Droplet DNA Amplification using a Novel "Tablet" Platform.
Angione S, Chauhan A, Tripathi A.
Abstract
We present a novel droplet based tablet platform for temporal polymerase chain reaction in microliter droplets. The simple design of the device does not require extensive processing or external equipment which allows for a greater ease of use and integration as a point-of-care diagnostic. We demonstrate its functionality to perform both Polymerase Chain Reaction (PCR) and reverse-transcription PCR for lambda 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 that 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 lambda phage PCR on our platform to be less than 2.0 copies/ul with an efficiency of 104.4% and similar sensitivity for a reverse-transcription PCR for influenza H3 RNA.
PMID:
22320164
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22320164
Real Time Droplet DNA Amplification using a Novel "Tablet" Platform.
Angione S, Chauhan A, Tripathi A.
Abstract
We present a novel droplet based tablet platform for temporal polymerase chain reaction in microliter droplets. The simple design of the device does not require extensive processing or external equipment which allows for a greater ease of use and integration as a point-of-care diagnostic. We demonstrate its functionality to perform both Polymerase Chain Reaction (PCR) and reverse-transcription PCR for lambda 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 that 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 lambda phage PCR on our platform to be less than 2.0 copies/ul with an efficiency of 104.4% and similar sensitivity for a reverse-transcription PCR for influenza H3 RNA.
PMID:
22320164
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/22320164