Over a decade ago, real-time PCR set the standard for sensitive DNA quantification with researchers relying heavily on the process for disease research, diagnosis, and preventative medicine. Today, digital PCR is poised to make a significant impact in the diagnostics field. By allowing for detection of nucleic acids at higher resolution and lower target levels, digital PCR has the ability to identify diseases earlier in progression, providing an advantage for diagnostics and preventative medicine. Its ability to precisely analyze nucleic acids at a molecular level makes it ideal for tasks such as detecting copy number variation and point mutations. This meeting will evaluate novel digital PCR technologies for research, demonstrate innovative amplification techniques, and present strategies for solving common research bottlenecks.

Main Conference

DIGITAL PCR AND EXISTING METHODS

Digital PCR: Moving Out of the Lab and onto the Bench

N. Reginald Beer, Ph.D., Medical Diagnostics Initiative Leader, Center for Micro and Nanotechnologies, Lawrence Livermore National Laboratory

Digital PCR provides extremely accurate quantitation as compared to traditional standard curve methods but at what cost? Originally, dPCR instruments were slow, time-consuming, proprietary, and expensive – all of which hindered the adoption of the technique to all but academic applications. Recently, the marketplace has begun to catch up – with only familiarity and the cost of operation hindering further adoption. In this talk we will discuss the benefits of dPCR, its most appropriate applications, and future trends driving the field.

Microfluidic Tools for PCR and Digital PCR

Bruce K. Gale, Ph.D., Associate Professor and Director, State of Utah Center of Excellence for Biomedical Microfluidics, Department of Mechanical Engineering, University of Utah

Microfluidics has been used to develop a rapid and specific PCR and digital PCR tools. This presentation will specifically address PCR methods such as gradient-based, continuous flow PCR and disk based digital PCR. These tools have been used to complete PCR and digital PCR with high resolution melting analysis in less than 8 minutes. Results of combining these systems with automated microfluidic extraction systems will also be presented.

Microreactor Arrays for Digital PCR, Sequencing, and Single Cell Analysis

Peter A. Sims, Ph.D., Assistant Professor and Principal Investigator, Columbia University Medical Center

PANEL: Implementing Digital PCR in the Lab: Practical Considerations

Panelists: Darren R. Link, Ph.D., Co-Founder and Vice President, R&D, Raindance Technologies, Inc.

George Karlin-Neumann, Ph.D., Sr. Director of ddPCR Assay Development, Bio-Rad Digital Biology Center

Additional Panelists to be Announced

(Sponsorship Opportunities Available)

DIGITAL DETECTION APPLICATIONS IN CANCER

Digital Detection of Ultra-Rare Nuclear and Mitochondrial Mutations: Fundamental and Clinical Application in Cancer and Disease

Jason H. Bielas, Ph.D., Assistant Member, Molecular Diagnostics Program, Fred Hutchinson Cancer Research Center (FHCRC); Affiliate Assistant Professorship, Department of Pathology, University of Washington

Droplet PCR for Personalized Cancer Healthcare

Pierre Laurent-Puig, M.D., Ph.D., Descartes University

Tumor cells are characterized by the presence of specific gene alterations, which can be useful for the monitoring of cancer patients through the development of biomarkers such as a free circulating tumor DNA in different body fluids. Such an approach led us to the development of highly sensitive methods because the tumor DNA is buried in an excess of normal DNA. The digital droplet PCR, enabled us to reach this goal through an easy and reproducible method, which had been validated in a series of metastatic colorectal cancer patients for KRAS mutations.

DNA Methylation Analysis Using Digital MethyLight Technology

Daniel Weisenberger, Ph.D., Assistant Professor of Research and Laboratory, USC Epigenome Center

DNA methylation changes are frequently observed in all human cancers, and can be also identified in patient blood. Here we describe the development of Digital MethyLight technology, a digital PCR application of MethyLight, sensitively detecting DNA methylation in primary tissues and fluids. We have identified and quantitated single-molecule, cancer-specific DNA hypermethylation events in plasma and serum samples from patients with cancer. This technology has promise in cancer detection and surveillance.

DIGITAL DETECTION APPLICATIONS IN PRENATAL DIAGNOSTICS

Digital Amplification of DNA Molecules and Metaphase Chromosomes with Applications in Prenatal Diagnosis

Christina Fan, Ph.D., Senior Scientist, ImmuMetrix LLC

This presentation will cover our work in developing the application of microfluidic digital PCR for aneuploidy detection, and the extension of digital PCR of DNA molecules to microfluidic digital multiple strand displacement amplification (MDA) of metaphase chromosomes from single cells. Digital MDA of chromosomes enables whole-genome haplotyping of human individuals, and the application in noninvasive prenatal diagnosis would be also be described.

PANEL: NGS or Digital PCR?

• Will sequencing technology mature to the point where digital PCR is unnecessary?
• Is there room for both technologies?
• How can the two work in tandem?
• Will cost of these technologies be prohibitive?

THE NEXT WAVE OF DIGITAL DEVICES

Large-Scale Droplet Arrays for Digital Microfluidics and Digital PCR

Abraham P. Lee, Ph.D., William J. Link Professor and Chair, Department of Biomedical Engineering; Director, Micro/Nano Fluidics Fundamentals Focus (MF3) Center, University of California, Irvine

Digital PCR is becoming a powerful tool in digital biology applications such as single-cell analysis and single-copy nucleic acid detection. In order to increase the dynamic range of digital PCR, a larger number of droplet (emulsion) reactors are required to detect a wider range of DNA concentrations. In this presentation, a large-scale (up to 1-million) droplet array is described that is enabled by droplet microfluidics that also enables monodispersed reactor volumes and stacking in 3-D for higher droplet density per unit area. In addition, our platform enables nucleic acid processing with real-time fluorescence measurements, which provide temporal information about PCR amplification.

Real-Time Droplet DNA Amplification with a New Tablet Platform

Anubhav Tripathi, Ph.D., Co-Director, Center for Biomedical Engineering, Brown University

Digital Nucleic Acid Amplification in a Sample Self-Digitization (SD) Chip

Daniel T. Chiu, Ph.D., A. Bruce Montgomery Professor of Chemistry, Endowed Professor in Analytical Chemistry, University of Washington

This presentation will describe the development and operation of our sample self-digitization (SD) chip and its application in digital nucleic acid amplification. Specifically, I will describe the realization of digital LAMP in the SD chip as well as the implementation of digital PCR using the SD chip.