Sequence specific sorting of DNA molecules with FACS using 3dPCR

Abstract

Genetic heterogeneity is an important feature of many biological systems, but introduces technical challenges to their characterization. Even with the best modern instruments, only a small fraction of DNA molecules present in a sample can be read, and they are recovered in the form of short, hundred-base reads. In this paper, we introduce 3dPCR, a method to sort DNA molecules with sequence specificity. 3dPCR allows heterogeneous populations of DNA to be sorted to recover long targets for deep sequencing. It is valuable whenever a target sequence is rare in a mixed population, such as for characterizing mutations in heterogeneous cancer cell populations or identifying cells containing a specific genetic sequence or infected with a target virus.

Precision public health to inhibit the contagion of disease and move toward a future in which microbes spread health

“…The usefulness of spatial and temporal metadata depends on how much they reveal heterogeneity. PCR can now be carried out inside nanoliter or picoliter volumes with many millions carried out in parallel and rare hits separated by Florescence Activated Cell Sorting (FACS) from a majority of negative droplets [25]. Single molecule sequencing is becoming routine and molecules can uniquely labeled with barcoded primers.…”

Droplet microfluidics for synthetic biology

“…6,7 Continuous methods can rapidly generate monodisperse droplets (up to 1-10 kHz), and are especially useful for high-throughput screening applications. 8,9 However, they allow limited control over addition or subtraction of reagents once droplets are formed. In contrast, digital droplet formation methods, while offering lower-throughput, provide ondemand droplet manipulation and control.…”

Finding a helix in a haystack: nucleic acid cytometry with droplet microfluidics

“…A recent demonstration of cell-free DNA sorting enriched for Lambda virus genomes in a background of ΦX174, and used double emulsions to sort using FACS. 36 A similar workflow can be applied to virus that establish latent reservoirs.…”

“…It is possible to couple the advantages of droplets with the ease of use and availability of fluorescence-activated cell sorting (FACS) instruments using double emulsions. 36,78,101 These water-in-oil-in-water (W/O/W) emulsions can be loaded onto FACS instruments directly. FACS is a mature technology and instruments are often located in core facilities where they are calibrated daily and well maintained.…”