Lyle Richard Middendorf

The challenges of sequencing by synthesis

DNA sequencing-by-synthesis (SBS) technology, using a polymerase or ligase enzyme as its core biochemistry, has already been incorporated in several second-generation DNA sequencing systems with significant performance. Notwithstanding the substantial success of these SBS platforms, challenges continue to limit the ability to reduce the cost of sequencing a human genome to

Correlation between Scattering Properties of Silver Particle Arrays and Fluorescence Enhancement

100,000 or less. Achieving dramatically reduced cost with enhanced throughput and quality will require the seamless integration of scientific and technological effort across disciplines within biochemistry, chemistry, physics and engineering. The challenges include sample preparation, surface chemistry, fluorescent labels, optimizing the enzyme-substrate system, optics, instrumentation, understanding tradeoffs of throughput versus accuracy, and read-length/phasing limitations. By framing these challenges in a manner accessible to a broad community of scientists and engineers, we hope to solicit input from the broader research community on means of accelerating the advancement of genome sequencing technology.

Versatile infrared laser scanner/electrophoresis apparatus

We report on the nanofabrication of patterned silver particle arrays using electron-beam lithography and the evaluation of their optical properties using backscattering and fluorescence spectroscopy. The silver particles varied in size from 100 to 250 nm and were in the shape of circles, squares, and triangles. Three inter-particle separations, 40, 65, and 90 nm as measured from the side of one particle to the side of the next particle, were used. We observed distinctive patterns of backscattering and fluorescence intensity depending on the particle size, inter-particle spacing, and excitation/emission wavelength used. Our approach allows for a study of the correlation between the backscattering intensities and fluorescence enhancement of silver particle arrays, which can be used to optimize the arrays for multi-fluorophore configuration for advanced sensing designs.

Near-Infrared Fluorescence Instrumentation for DNA Analysis

An infrared fluorescence microscope consisting of a laser diode for exciting infrared fluorophores attached to DNA oligo-nucleotides and a silicon avalanche photodiode for detecting the infrared emission has been designed. The microscope was mounted on a scanning platform which could be optimally focused on an electrophoretic gel (0.1 - 0.4 mm thick) sandwiched between two glass plates. Background fluorescence is minimal in the infrared region of the optical spectrum. In addition, the optics were designed to further minimize this background fluorescence while maximizing the signal output. A 5 pM fluorophore-DNA concentration in unpolymerized gel solution (about 2000 molecules in an irradiated volume of 600 pL) gave a signal-to-noise ratio of 4:1, 3:1, and 2:1 for a glass-gel-glass sandwich made using quartz, borosilicate, and soda-lime glass, respectively.

Near-Infrared, Surface-Enhanced Fluorescence Using Silver Nanoparticle Aggregates in Solution

Information throughput relating to DNA analysis is enhanced by increasing sample channels (e.g., geometric, spectral, temporal discrimination), increasing information per sample channel (e.g., sequence read length, use of multiplexed loci), decreasing redundant information among samples, and decreasing time of sample preparation, data collection and processing. Recently, the use of solid-state optical components has provided a highly sensitive, yet economical detection system based on near-infrared (NIR) fluorescence. This is important for enhancing throughput for both electrophoresis-based automated analysis as well as for analysis using biochips and high density gridding.

Two-dimensional infrared fluorescence scanner used for DNA analysis

Fluorescence spectroscopy is used in many life science and clinical research diagnostic assays. Improvements in the sensitivity and limit-of-detection of these assays may have profound implications. Here, we demonstrate a near-infrared, surface-enhanced fluorescence technology that increases the signal of IRDye 800CW-labeled streptavidin by up to 2,530-fold while improving the limit-of-detection 1,000-fold. Citrate-stabilized, silver nanoparticles that aggregate in solution were used with the dye-protein conjugate to form plasmon-active nanostructures. The technique is straightforward to implement and fully compatible with commercially available immunoassay instrumentation and consumables.

Imaging of single-chromophore molecules in aqueous solution near a fused-silica interface

A LI-COR Model 4000 DNA Sequencer has been modified by removing the internal scanning infrared fluorescence microscope and combining it with an external, orthogonal scanner. Due to the reduced background fluorescence and light scattering of nylon membranes in the near- infrared (8000 nm) as compared to the visible region of the optical spectrum, sensitivity of labeled DNA fragments is enhanced. Dot blots of dilution series of labeled oligonucleotides reveal a detection limit of 25 attomole (25 X 10-18 mole). DNA fragments blotted onto nylon membranes using direct transfer electrophoresis in multiplex DNA sequencing can also be detected and subsequently analyzed.

Time-resolved photon counting for multiplexed DNA electrophoreses separations

Single molecules of unconjugated Bodipy-Texas Red (BTR), BTR-dimer, and BTR conjugated to cysteine, in aqueous solutions are imaged using total-internal-reflection excitation and through-sample collection of fluorescence onto an intensified CCD camera, or a back-illuminated frame transfer CCD. The sample excitation is provided by the beam from a continuous-wave krypton ion laser, or a synchronously-pumped dye laser, operating at 568 nm. In order to essentially freeze molecular motion due to diffusion and thereby enhance image contrast, the laser beam is first passed through a mechanical shutter, which yields a 3-millisecond laser exposure for each camera frame. The laser beam strikes the fused-silica/sample interface at an angle exceeding the critical angle by about 1 degree. The resultant evanescent wave penetrates into the sample a depth of approximately 0.3 microns. Fluorescence from the thin plane of illumination is then imaged onto the camera by a water immersion apochromat (NA 1.2, WD 0.2mm). A Raman notch filter blocks Rayleigh and specular laser scatter and a band-pass-filter blocks most Raman light scatter that originates from the solvent. Single molecules that have diffused into the evanescent zone at the time of laser exposure yield near-diffraction-limited Airy disk images with diameters of ~5 pixels. While most molecules diffuse out of the evanescent zone before the next laser exposure, stationary or slowly moving molecules persisting over several frames, and blinking of such molecules are occasionally observed.

Continuous, On-Line, Real Time DNA Sequencing Using Multifluorescently Tagged Primers

Monte Carlo simulations are used to evaluate time resolved photon counting as a means for identifying and quantifying overlapping electropherogram bands, which have been labeled by dyes with disparate fluorescence lifetimes.

Continuous, on‐line DNA sequencing using a versatile infrared laser scanner/electrophoresis apparatus

DNA sequencing is a relatively new technique, just over a decade old. Scientifically, it is a very important technique because the DNA sequence gives us the ultimate resolution of the genetic material to the single base level and thus its complete information.