University of California

The United States of America As Represented by the United States

The United States of America

James H Jett

John C Martin

Richard A Keller

Peter M Goodwin

Babetta L Marrone

L Scott Cram

Mark L Hammond

Mary C Roslaniec

E Brooks Shera

W Patrick Ambrose

Daniel J Simpson

George C Saunders

W Kevin Grace

Robert K Moyzis

Robert L Ratliff

Richard G Posner

Alan Van Orden

Carleton C Stewart

Alan K Van Orden

Nicholas P Machara

Bill F Bentley

Apparatus and method enable imaging multiple fluorescent sample particles in a single flow channel. A flow channel defines a flow direction for samples in a flow stream and has a viewing plane perpendicular to the flow direction. A laser beam is formed as a ribbon having a width effective to cover the viewing plane. Imaging optics are arranged to view the viewing plane to form an image of the fluorescent sample particles in the flow stream, and a camera records the image formed by the imaging optics.

High throughput analysis of samples in flowing liquid

Single fluorescent molecules in a flowing sample stream are distinguished and identified using only a single laser excitation wavelength. A sample stream is formed containing a dilute mixture of single molecule fluorophores, wherein each one of the fluorophores is serially ordered in the sample stream. The sample stream is illuminated with s single excitation wavelength laser effective to excite each fluorophore one at a time. Fluorescence emission photons from each said fluorophore are detected. A burst size is determined for each fluorophore to identify each fluorophore. A pulsed laser may be used, where burst size and an intra-burst fluorescence decay rate for each fluorophore are determined simultaneously from the detected fluorescence emission photons. The burst size and the decay rate are correlated to identify each fluorophore.

Single molecule identification using selected fluorescence

Optical selection and collection of DNA fragments. The present invention includes the optical selection and collection of large (&gt;.mu.g) quantities of clonable, chromosome-specific DNA from a sample of chromosomes. Chromosome selection is based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Although more general procedures may be envisioned, the invention is demonstrated by processing chromosomes in a conventional flow cytometry apparatus, but where no droplets are generated. All chromosomes in the sample are first stained with at least one fluorescent analytic dye and bonded to a photochemically active species which can render chromosomal DNA unclonable if activated. After passing through analyzing light beam(s), unwanted chromosomes are irradiated using light which is absorbed by the photochemically active species, thereby causing photoinactivation. As desired chromosomes pass this photoinactivation point, the inactivating light source is deflected by an optical modulator; hence, desired chromosomes are not photoinactivated and remain clonable. The selection and photoinactivation processes take place on a microsecond timescale. By eliminating droplet formation, chromosome selection rates 50 times greater than those possible with conventional chromosome sorters may be obtained. Thus, usable quantities of clonable DNA from any source thereof may be collected.

Optical selection and collection of DNA fragments

A method is provided for sizing DNA fragments using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA piece or the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is linearly related to the fragment length. The distribution of DNA fragment sizes forms a characterization of the DNA piece for use in forensic and research applications.

DNA fragment sizing and sorting by laser-induced fluorescence

Method for rapid-base sequencing in DNA and RNA with two-base labeling and employing fluorescent detection of single molecules at two wavelengths. Bases modified to accept fluorescent labels are used to replicate a single DNA or RNA strand to be sequenced. The bases are then sequentially cleaved from the replicated strand, excited with a chosen spectrum of electromagnetic radiation, and the fluorescence from individual, tagged bases detected in the order of cleavage from the strand.

Method for rapid base sequencing in DNA and RNA with two base labeling

Method and apparatus for removing material from a gas. A mist created by a piezoelectric ultrasonic transducer is contacted with the gas and both gas and mist are passed through baffled separators. Liquid effluent from the separators contains solid material removed from the gas and gaseous material which reacted with the liquid or was absorbed by the liquid. The invention is useful for collecting a sample of material in a gas, such as a vapor in the atmosphere, and in cleaning a gas. A relatively concentrated solution of a material present in a gas in a very small concentration can be obtained.

Compact air scrubber

A method is provided for the rapid base sequencing of DNA or RNA fragments wherein a single fragment of DNA or RNA is provided with identifiable bases and suspended in a moving flow stream. An exonuclease sequentially cleaves individual bases from the end of the suspended fragment. The moving flow stream maintains the cleaved bases in an orderly train for subsequent detection and identification. In a particular embodiment, individual bases forming the DNA or RNA fragments are individually tagged with a characteristic fluorescent dye. The train of bases is then excited to fluorescence with an output spectrum characteristic of the individual bases. Accordingly, the base sequence of the original DNA or RNA fragment can be reconstructed.

Method for rapid base sequencing in DNA and RNA

The disclosure is directed to an apparatus for eliminating background interference during fluorescence measurements in a multiple laser flow cytometer. A biological particle stained with fluorescent dyes is excited by a laser. A fluorescence detector detects the fluorescence. The particle scatters light and a gate signal is generated and delayed until the biological particle reaches the next laser. The delayed signal turns on this next laser, which excites a different stained component of the same biological particle.

Apparatus for eliminating background interference in fluorescence

Growing community of inventors

Los Alamos, NM, United States of America

James H Jett