Droplet-Based Microfluidic Systems for High-Throughput Single DNA Molecule Isothermal Amplification and Analysis

被引：212

作者：

Mazutis, Linas ^{[1
]}

Araghi, Ali Fallah ^{[1
]}

Miller, Oliver J. ^{[1
]}

Baret, Jean-Christophe ^{[1
]}

Frenz, Lucas ^{[1
]}

Janoshazi, Agnes ^{[1
,3
]}

Taly, Valerie ^{[1
]}

Miller, Benjamin J. ^{[2
]}

Hutchison, J. Brian ^{[2
]}

Link, Darren ^{[2
]}

Griffiths, Andrew D. ^{[1
]}

Ryckelynck, Michael ^{[1
]}

机构：

[1] Univ Strasbourg, CNRS, ISIS, UMR 7006, F-67083 Strasbourg, France

[2] RainDance Technol Inc, Lexington, MA 02421 USA

[3] Univ Strasbourg, IGBMC, CNRS, UMR 7104, F-67404 Illkirch Graffenstaden, France

来源：

ANALYTICAL CHEMISTRY | 2009年 / 81卷 / 12期

基金：

英国医学研究理事会;

关键词：

POLYMERASE-CHAIN-REACTION; PICOLITER DROPLETS; DIGITAL PCR; REAL-TIME; ON-CHIP; POLY(DIMETHYLSILOXANE); GALACTOSIDASE; EMULSIONS; EVOLUTION; PROTEIN;

D O I：

10.1021/ac900403z

中图分类号：

O65 [分析化学];

学科分类号：

070302 ; 081704 ;

摘要：

We have developed a method for high-throughput isothermal amplification of single DNA molecules in a droplet-based microfluidic system. DNA amplification in droplets was analyzed using an intercalating fluorochrome, allowing fast and accurate "digital" quantification of the template DNA based on the Poisson distribution of DNA molecules in droplets. The clonal amplified DNA in each 2 pL droplet was further analyzed by measuring the enzymatic activity of the encoded proteins after fusion with a 15 pL droplet containing an in vitro translation system.

引用

页码：4813 / 4821

页数：9

共 33 条

[1] Dielectrophoretic manipulation of drops for high-speed microfluidic sorting devices [J].

Ahn, K ;

Kerbage, C ;

Hunt, TP ;

Westervelt, RM ;

Link, DR ;

Weitz, DA .

APPLIED PHYSICS LETTERS, 2006, 88 (02) :1-3

[2] Electrocoalescence of drops synchronized by size-dependent flow in microfluidic channels [J].

Ahn, Keunho ;

Agresti, Jeremy ;

Chong, Henry ;

Marquez, Manuel ;

Weitz, D. A. .

APPLIED PHYSICS LETTERS, 2006, 88 (26)

[3]

AMBUEL Y, 2003, INNOVATIONS, V16, P3

[4] Fluorescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity [J].

Baret, Jean-Christophe ;

Miller, Oliver J. ;

Taly, Valerie ;

Ryckelynck, Michael ;

El-Harrak, Abdeslam ;

Frenz, Lucas ;

Rick, Christian ;

Samuels, Michael L. ;

Hutchison, J. Brian ;

Agresti, Jeremy J. ;

Link, Darren R. ;

Weitz, David A. ;

Griffiths, Andrew D. .

LAB ON A CHIP, 2009, 9 (13) :1850-1858

[5] On-chip single-copy real-time reverse-transcription PCR in isolated picoliter droplets [J].

Beer, N. Reginald ;

Wheeler, Elizabeth K. ;

Lee-Houghton, Lorenna ;

Watkins, Nicholas ;

Nasarabadi, Shanavaz ;

Hebert, Nicole ;

Leung, Patrick ;

Arnold, Don W. ;

Bailey, Christopher G. ;

Colston, Bill W. .

ANALYTICAL CHEMISTRY, 2008, 80 (06) :1854-1858

[6] On-chip, real-time, single-copy polymerase chain reaction in picoliter droplets [J].

Beer, N. Reginald ;

Hindson, Benjamin J. ;

Wheeler, Elizabeth K. ;

Hall, Sara B. ;

Rose, Klint A. ;

Kennedy, Ian M. ;

Colston, Bill W. .

ANALYTICAL CHEMISTRY, 2007, 79 (22) :8471-8475

[7] Droplet fusion by alternating current (AC) field electrocoalescence in microchannels [J].

Chabert, M ;

Dorfman, KD ;

Viovy, JL .

ELECTROPHORESIS, 2005, 26 (19) :3706-3715

[8] Microfluidic methods for generating continuous droplet streams [J].

Christopher, G. F. ;

Anna, S. L. .

JOURNAL OF PHYSICS D-APPLIED PHYSICS, 2007, 40 (19) :R319-R336

[9] An integrated device for monitoring time-dependent in vitro expression from single genes in picolitre droplets [J].

Courtois, Fabienne ;

Olguin, Luis F. ;

Whyte, Graeme ;

Bratton, Daniel ;

Huck, Wilhelm T. S. ;

Abell, Chris ;

Hollfelder, Florian .

CHEMBIOCHEM, 2008, 9 (03) :439-446

[10] Nucleic acid amplification of individual molecules in a microfluidic device [J].

Dettloff, Roger ;

Yang, Esther ;

Rulison, Aaron ;

Chow, Andrea ;

Farinas, Javier .

ANALYTICAL CHEMISTRY, 2008, 80 (11) :4208-4213

← 1 2 3 4 →