Molecular Foundry Seminar

"Target Sequence Detection Method for Single DNA Molecules via Extension Flow at a Stagnation Point"

Rebecca Dylla-Spears, UC Berkeley,

Tuesday, November 17 at 1:30 pm, Room 67-3111

Abstract:

We have developed a single-molecule method enabling the direct detection of widely spaced DNA target sequences over genomic lengths of DNA.  First, we have created a fluorescent marker using a mutant EcoRI restriction endonuclease (K249C) that enables prolonged, direct visualization of specific sequences on genomic lengths of double-stranded (ds) DNA. Biotinylated enzyme is incubated with dsDNA and NeutrAvidin-coated, fluorescent nanospheres under conditions that allow enzyme binding but prevent cleavage. We demonstrate the marker’s ability to bind specifically to its target sequence using both bulk gel-shift assays and single-molecule methods. 

Next, we use a microfluidic stagnation point flow--specifically planar extensional flow in a cross slot--to trap and extend single marker-DNA complexes into a linear conformation for detection of target sequences along the DNA backbone. The degree of DNA elongation can be controlled using the flow strength (i.e., a non-dimensional flow rate) in the device.  Both the DNA backbone and the bound markers are observed directly using fluorescence microscopy, and the location of the markers along the DNA backbone is measured. We find that our method permits detection of each of the five expected target site positions to within 1.5 kb with standard deviations of <1.5 kb.  We compare the method’s precision and accuracy at molecular extensions of 68% and 88% of the contour length to binding distributions from similar data obtained via slide immobilization.