| Autor: |
Griffis Joshua, Gene Malkin, Andrew Nicholson, Shilpi P. Vyas, Robert H. Meltzer, Mikhail M. Safranovitch |
| Jazyk: |
angličtina |
| Předmět: |
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| Zdroj: |
Biophysical Journal. (2):278a-279a |
| ISSN: |
0006-3495 |
| DOI: |
10.1016/j.bpj.2013.11.1630 |
| Popis: |
Fluorescent microscopic observation of DNA stretching in homogenous elongational flow has previously been used to characterize biophysical properties of the polymer. Typically, individual molecules are trapped at the stagnation point of opposed fluidic flows in a cross-slot microfluidic structure. Such observations intrinsically have a low throughput of analyzable molecules. We present a continuous-flow microfluidic funnel that replicates the intramolecular tension distribution established in cross-slot DNA stretching experiments, but achieves greater than 10 megabase per second analyzable DNA throughput.Intercalating fluorescent dyes are required for single-molecule visualization of stretched DNA. DNA elasticity under tension however is affected by bound intercalator. Careful experimental control of the relative concentration of intercalator and DNA is therefore required for reproducible DNA stretching. In the presented device, intercalator is introduced using convergent sheathing flows, which center DNA in the microfunnel over a series of confocal laser excitation spots. The intercalation reaction therefore proceeds one molecule of DNA at a time, thus eliminating dependence on DNA concentration. Using this experimental platform, we demonstrate differing effects on DNA elasticity for the monomeric dye POPRO-1 and its bis-intercalating analogue POPO-1. We also characterize the effect of intramolecular tension on the orientation of intercalating dye in its DNA binding site using single-molecule fluorescence anisotropy.Better understanding of stretching mechanics and tension distribution along extended DNA yields practical improvements in comparing fluorescence traces of site-specific probes to sequence-derived templates. This enhances genomic comparison and identification in continuous flow optical mapping applications. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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