title: High-performance single cell genetic analysis using microfluidic emulsion generator arrays.
abstract: High-throughput genetic and phenotypic analysis at the single cell level is critical to advance our understanding of the molecular mechanisms underlying cellular function and dysfunction. Here we describe a high-performance single cell genetic analysis (SCGA) technique that combines high-throughput microfluidic emulsion generation with single cell multiplex polymerase chain reaction (PCR). Microfabricated emulsion generator array (MEGA) devices containing 4, 32, and 96 channels are developed to confer a flexible capability of generating up to 3.4 x 10(6) nanoliter-volume droplets per hour. Hybrid glass-polydimethylsiloxane diaphragm micropumps integrated into the MEGA chips afford uniform droplet formation, controlled generation frequency, and effective transportation and encapsulation of primer functionalized microbeads and cells. A multiplex single cell PCR method is developed to detect and quantify both wild type and mutant/pathogenic cells. In this method, microbeads functionalized with multiple forward primers targeting specific genes from different cell types are used for solid-phase PCR in droplets. Following PCR, the droplets are lysed and the beads are pooled and rapidly analyzed by multicolor flow cytometry. Using Escherichia coli bacterial cells as a model, we show that this technique enables digital detection of pathogenic E. coli O157 cells in a high background of normal K12 cells, with a detection limit on the order of 1/10(5). This result demonstrates that multiplex SCGA is a promising tool for high-throughput quantitative digital analysis of genetic variation in complex populations.
authors.affiliation: Department of Chemistry, University of California, Berkeley, California 94720, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zeng
authors.forename: Richard
authors.initials: R
authors.lastname: Novak
authors.forename: Joe
authors.initials: J
authors.lastname: Shuga
authors.forename: Martyn T
authors.initials: MT
authors.lastname: Smith
authors.forename: Richard A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification; D057166:High-Throughput Screening Assays; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction