+ Site Statistics
+ Search Articles
+ PDF Full Text Service
How our service works
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ Translate
+ Recently Requested

Single-molecule DNA amplification and analysis in an integrated microfluidic device



Single-molecule DNA amplification and analysis in an integrated microfluidic device



Analytical Chemistry 73(3): 565-570



Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.

Please choose payment method:






(PDF emailed within 0-6 h: $19.90)

Accession: 047377108

Download citation: RISBibTeXText

PMID: 11217764

DOI: 10.1021/ac001026b


Related references

Droplet-based microfluidic systems for high-throughput single DNA molecule isothermal amplification and analysis. Analytical Chemistry 81(12): 4813-4821, 2009

Single molecule biology Direct RNA quantitation using single molecule counting in a microfluidic device to monitor gene expression. Journal of the American Society of Nephrology 14(Abstracts Issue): 24A, 2003

A microwell array device with integrated microfluidic components for enhanced single-cell analysis. Electrophoresis 30(24): 4166-4171, 2009

An integrated microfluidic device for DNA purification and PCR amplification of STR fragments. Forensic Science International. Genetics 4(3): 178-186, 2010

Microfluidic device for continuous single cells analysis via Raman spectroscopy enhanced by integrated plasmonic nanodimers. Optics Express 24(2): A180-A190, 2016

High-throughput microfluidic device for single cell analysis using multiple integrated soft lithographic pumps. Electrophoresis 37(10): 1337-1344, 2016

Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry. Analytical Chemistry 82(3): 967-973, 2010

Dynamic Generation of Concentration- and Temporal-Dependent Chemical Signals in an Integrated Microfluidic Device for Single-Cell Analysis. Analytical Chemistry 90(14): 8331-8336, 2018

Rapid PCR amplification using a microfluidic device with integrated microwave heating and air impingement cooling. Lab on a Chip 10(13): 1725-1728, 2010

Microfluidic Systems Integrated With a Sample Pretreatment Device for Fast Nucleic-Acid Amplification. Journal of Microelectromechanical Systems 17(2): 288-301, 2008

Development of a real-world direct interface for integrated DNA extraction and amplification in a microfluidic device. Lab on a Chip 11(3): 443-448, 2011

Microfluidic whole genome amplification device for single cell sequencing. Analytical Chemistry 86(19): 9386-9390, 2014

Single DNA molecule isolation and trapping in a microfluidic device. Chemphyschem 8(12): 1875-1880, 2007

Microfluidic DNA Stretching Device for Single-Molecule Diagnostics. Methods in Molecular Biology 1547: 105-111, 2017

Microfluidic device for single-molecule experiments with enhanced photostability. Journal of the American Chemical Society 131(38): 13610-2, 2009