33 research outputs found
Peering Below the Diffraction Limit: Robust and Specific Sorting of Viruses with Flow Cytometry
Background: Viruses are incredibly diverse organisms and impact all forms of life on Earth; however, individual virions are challenging to study due to their small size and mass, precluding almost all direct imaging or molecular analysis. Moreover, like microbes, the overwhelming majority of viruses cannot be cultured, impeding isolation, replication, and study of interesting new species. Here, we introduce PCR-activated virus sorting, a method to isolate specific viruses from a heterogeneous population. Specific sorting opens new avenues in the study of uncultivable viruses, including recovering the full genomes of viruses based on genetic fragments in metagenomes, or identifying the hosts of viruses.
Methods: PAVS enables specific sorting of viruses with flow cytometry. A sample containing a virus population is processed through a microfluidic device to encapsulate it into droplets, such that the droplets contain different viruses from the sample. TaqMan PCR reagents are also included targeting specific virus species such that, upon thermal cycling, droplets containing the species become fluorescent. The target viruses are then recovered via droplet sorting. The recovered virus genomes can then be analyzed with qPCR and next generation sequencing.
Results and Conclusions: We describe the PAVS workflow and demonstrate its specificity for identifying target viruses in a heterogeneous population. In addition, we demonstrate recovery of the target viruses via droplet sorting and analysis of their nucleic acids with qPCR
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Sequence specific sorting of DNA molecules with FACS using 3dPCR.
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
Sequence specific sorting of DNA molecules with FACS using 3dPCR.
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
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Patterning microfluidic device wettability with spatially-controlled plasma oxidation
Microfluidic devices can form double emulsions with uniform properties, but require cumbersome fabrication steps to pattern their wettability. We demonstrate spatially-controlled plasma oxidation to create wettability patterns for forming double emulsions. Our method performs comparably to chemical techniques but is simpler, more reliable, and scalable to patterning large arrays of drop makers
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Bulk double emulsification for flow cytometric analysis of microfluidic droplets
Droplet microfluidics is valuable for applications in chemistry and biology, but generates massive numbers of droplets that must be analyzed and sorted. Here, we describe a simple approach to bulk double emulsify microfluidic emulsions for analysis and sorting with commercial flow cytometers. We illustrate the method by using it to identify droplets based on nucleic acid content. Though simple, our method provides a general approach for analyzing and sorting microfluidic droplets without custom microfluidic double emulsifiers or sorters
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Observing Biosynthetic Activity Utilizing Next Generation Sequencing and the DNA Linked Enzyme Coupled Assay
Currently,
the identification of new genes drastically outpaces
current experimental methods for determining their enzymatic function.
This disparity necessitates the development of high-throughput techniques
that operate with the same scalability as modern gene synthesis and
sequencing technologies. In this paper, we demonstrate the versatility
of the recently reported DNA-Linked Enzyme-Coupled Assay (DLEnCA)
and its ability to support high-throughput data acquisition through
next-generation sequencing (NGS). Utilizing methyltransferases, we
highlight DLEnCA’s ability to rapidly profile an enzyme’s
substrate specificity, determine relative enzyme kinetics, detect
biosynthetic formation of a target molecule, and its potential to
benefit from the scales and standardization afforded by NGS. This
improved methodology minimizes the effort in acquiring biosynthetic
knowledge by tying biochemical techniques to the rapidly evolving
abilities in sequencing and synthesizing DNA
Rho kinase modulates postnatal adaptation of the pulmonary circulation through separate effects on pulmonary artery endothelial and smooth muscle cells
At birth, pulmonary vasodilation occurs concomitant with the onset of air-breathing life. Whether and how Rho kinase (ROCK) modulates the perinatal pulmonary vascular tone remains incompletely understood. To more fully characterize the separate and interactive effects of ROCK signaling, we hypothesized that ROCK has discrete effects on both pulmonary artery (PA): 1) endothelial cell (PAEC) nitric oxide (NO) production and contractile state; and 2) smooth muscle cell tone independent of endothelial NO synthase (eNOS) activity. To test these hypotheses, NO production and endothelial barrier function were determined in fetal PAEC under baseline hypoxia and following exposure to normoxia with and without treatment with Y-27632, a specific pharmacological inhibitor of ROCK. In acutely instrumented, late-gestation ovine fetuses, eNOS was inhibited by nitro-l-arginine infusion into the left PA (LPA). Subsequently, fetal lambs were mechanically ventilated (MV) with 100% oxygen in the absence (control period) and presence of Y-27632. In PAEC, treatment with Y-27632 had no effect on cytosolic calcium but did increase normoxia-induced NO production. Moreover, acute normoxia increased PAEC barrier function, an effect that was potentiated by Y-27632. In fetal lambs, MV during the control period had no effect on LPA flow. In contrast, MV after Y-27632 increased LPA flow and fetal arterial Po2 (PaO2) and decreased PA pressure. In conclusion, ROCK activity modulates vascular tone in the perinatal pulmonary circulation via combined effects on PAEC NO production, barrier function, and smooth muscle tone. ROCK inhibition may represent a novel treatment strategy for neonatal pulmonary vascular disease