Bio-rad Laboratories, Inc.

Raindance Technologies, Inc.

Lariat Biosciences, Inc.

Other

Jonathan William Larson

Darren Roy Link

Qun Zhong

Yevgeny Yurkovetsky

Mark Weary

Benjamin J Miller

Andrew Wilson

Brian Hutchison

Alexandra Damiano

The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.

Digital analyte analysis

The invention provides methods for assessing one or more predetermined characteristics or properties of a microfluidic droplet within a microfluidic channel, and regulating one or more fluid flow rates within that channel to selectively alter the predetermined microdroplet characteristic or property using a feedback control.

Manipulation of microfluidic droplets

In the invention described here, the conventional need for feedback control is eliminated by a passive, open-loop approach using a novel microfluidic droplet generator with a step enhancement. The invented droplet generator yields uniform droplet volumes over a wide range of operating pressures, delivering robust performance at a very low cost. The invention also describes a method of droplet generation whereby the step enhancement improves the performance of any squeeze-mode or dripping-mode droplet generator, including but not limited to bridge-mode and flow-focusing configurations. The performance of the invention is sufficiently stable that it can be operated manually yet still deliver best-in-class microfluidic performance. Thus, not only does the invention greatly simplify and reduce the cost of operation in the laboratory, it opens the possibility of performing precision biology out in the field and off of the electrical grid.

Microfluidic device

The invention generally relates to methods for forming mixed droplets. In certain embodiments, methods of the invention involve forming a droplet, and contacting the droplet with a fluid stream, wherein a portion of the fluid stream integrates with the droplet to form a mixed droplet.

Methods for forming mixed droplets

Techniques are provided for generating, manipulating, and measuring fluidic droplets in mixed phase systems based on establishing transient continuities between otherwise spatially separated phases. In certain methods of the invention, electrodes in contact with the continuous phases allow electrical monitoring of continuity or proximity of separated phases as a means to characterize droplets. In other methods of the invention, fluidic continuity provides a means for generating droplets, injecting or extracting the contents of droplets, and sorting droplets. Chemical techniques are also provided that use these droplet-based methods, or others, to quantify and identify nucleic acids through incorporation into hydrogel particles. The nucleic acids are entrapped either actively by chemical incorporation during gel polymerization or passively by chain entanglement. After incorporation into the hydrogel particles, the nucleic acids are solvent accessible either at the particle periphery or within internal pores for further biochemical manipulations and characterization. In one important aspect, the invention combines the high specificity, high sensitivity, and unbiased performance of clonal DNA amplification in free solution with the simplicity of permanently co-localizing the separate reaction products onto rigid substrates for myriad biochemical applications.

Growing community of inventors

Chelsea, MA, United States of America

Jonathan William Larson