High throughput nucleic acid profiling of single cells

Abstract

Methods of profiling the nucleic acid composition of single cells and tools for same. The methods can include isolating a single cell in a liquid droplet, lysing the single cell in the liquid droplet to release template nucleic acid from the cell, amplifying the template nucleic acid in the liquid droplet to generate amplified nucleic acid, and detecting the amplified nucleic acid in the liquid droplet. The methods can be useful for profiling expression patterns and / or detecting genetic characteristics such as single nucleotide polymorphisms. The tools include nucleic acid logic gates, including polymerase-dependent logic gates. The logic gates can perform logical operations such as YES, NOT, AND, OR, AND-NOT, NOT-AND, NOT-OR, EXCLUSIVE-OR, EXCLUSIVE-NOR, and IMPLY. The tools also include microfluidic systems for performing the methods.

Description

FIELD OF THE INVENTION[0001]The invention is directed to high-throughput methods, systems, and devices for profiling the nucleic acid composition of single cells within a heterogeneous population of cells.BACKGROUND[0002]Cellular heterogeneity and its impact on biological function and disease are becoming increasingly important for questions in human immunology, stem cell biology, and cancer research. By transcriptionally analyzing individual cells with reverse-transcriptase polymerase chain reaction (RT-PCR), for example, it is possible to identify rare cells or transient cell states that are unobservable when studying the entire population in bulk (Bendall et al. 2012, Kalisky and Blainey et al. 2011, Kalisky and Quake 2011, Levsky et al. 2003). However, obtaining meaningful information on these cells necessitates tools capable of high-throughput analysis. Current methods for manipulating, isolating, and transcriptionally profiling single cells with RT-PCR are cumbersome and limit...

AI Technical Summary

Benefits of technology

This approach allows for the efficient profiling of nucleic acid composition of large numbers of cells, achieving high sensitivity and specificity, and enabling the detection of rare cell types like circulating tumor cells with high throughput.

Problems solved by technology

Current methods for manipulating, isolating, and transcriptionally profiling single cells with RT-PCR are cumbersome and limited in throughput, enabling the examination of just hundreds of individual cells.

However, an obstacle to realizing the potential of this approach is that, at concentrations of a single cell in a microdroplet, cell lysate is a potent inhibitor of RT-PCR (Arezi et al.

Methods using large droplets have only been able to analyze ˜100 cells in total, while the agarose method is unable to use TaqMan probes or cell staining, precluding correlation of specific cell types with associated transcriptional targets.

However, because each microwell and its control valves must be fabricated and individually controlled, throughput is also limited to just a few hundred cells in total (Kalisky and Blainey et al.

While this dilution approach is effective, it is a highly cumbersome process that limits processivity.

Images