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Title: On-chip real-time single-copy polymerase chain reaction in picoliter droplets

Abstract

The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10{sup 6} smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of {approx}18, twenty cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
929191
Report Number(s):
UCRL-JRNL-230233
TRN: US200815%%231
DOE Contract Number:  
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Analytical Chemistry, vol. 79, no. 22, November 15, 2007, pp. 8471-8475; Journal Volume: 79; Journal Issue: 22
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL AND ANALYTICAL CHEMISTRY; 47 OTHER INSTRUMENTATION; AMPLIFICATION; DETECTION; DNA; FLUORESCENCE; NUCLEIC ACIDS; POLYMERASE CHAIN REACTION; SILICON; STATISTICS

Citation Formats

Beer, N R, Hindson, B, Wheeler, E, Hall, S B, Rose, K A, Kennedy, I, and Colston, B. On-chip real-time single-copy polymerase chain reaction in picoliter droplets. United States: N. p., 2007. Web. doi:10.1021/ac701809w.
Beer, N R, Hindson, B, Wheeler, E, Hall, S B, Rose, K A, Kennedy, I, & Colston, B. On-chip real-time single-copy polymerase chain reaction in picoliter droplets. United States. doi:10.1021/ac701809w.
Beer, N R, Hindson, B, Wheeler, E, Hall, S B, Rose, K A, Kennedy, I, and Colston, B. Fri . "On-chip real-time single-copy polymerase chain reaction in picoliter droplets". United States. doi:10.1021/ac701809w. https://www.osti.gov/servlets/purl/929191.
@article{osti_929191,
title = {On-chip real-time single-copy polymerase chain reaction in picoliter droplets},
author = {Beer, N R and Hindson, B and Wheeler, E and Hall, S B and Rose, K A and Kennedy, I and Colston, B},
abstractNote = {The first lab-on-chip system for picoliter droplet generation and PCR amplification with real-time fluorescence detection has performed PCR in isolated droplets at volumes 10{sup 6} smaller than commercial real-time PCR systems. The system utilized a shearing T-junction in a silicon device to generate a stream of monodisperse picoliter droplets that were isolated from the microfluidic channel walls and each other by the oil phase carrier. An off-chip valving system stopped the droplets on-chip, allowing them to be thermal cycled through the PCR protocol without droplet motion. With this system a 10-pL droplet, encapsulating less than one copy of viral genomic DNA through Poisson statistics, showed real-time PCR amplification curves with a cycle threshold of {approx}18, twenty cycles earlier than commercial instruments. This combination of the established real-time PCR assay with digital microfluidics is ideal for isolating single-copy nucleic acids in a complex environment.},
doi = {10.1021/ac701809w},
journal = {Analytical Chemistry, vol. 79, no. 22, November 15, 2007, pp. 8471-8475},
number = 22,
volume = 79,
place = {United States},
year = {Fri Apr 20 00:00:00 EDT 2007},
month = {Fri Apr 20 00:00:00 EDT 2007}
}