University of Massachusetts

Becton Dickinson and Company Limited

Robert H Singer

Jeanne B Lawrence

Krishan L Taneja

Joan C Politz

Kenton L Lohman

Carol Villnave Johnson

Andrea M Femino

Jean Marie Mathys

Edward H Kislauskis

Improved methodologies for in-situ hybridization and non-isotopic detection of nucleic acid sequences are provided which offer major increases of resolution, sensitivity, and simplicity unavailable in previously known techniques. The methodology is able to detect even a single-copy of a specific nucleic acid of interest under controlled conditions regardless of whether these are DNA or RNA sequences; or whether the nucleic acid sequence of interest is localized in the chromosomes, nucleus, or cytoplasm of a cell. The methods employ a variety of non-isotopic labels and detection means for rapid and reliable assays. The invention is also provided in kit form for use in the clinical/diagnostic laboratory such that a relatively unskilled person can accurately and reproducibly detect even a single-copy of a specific nucleic acid of interest.

In-situ hybridization of single-copy and multiple-copy nucleic acid sequences

Non-isotopic in-situ hybridization method for detection of nucleic acids

Disclosed is a method of detecting a trinucleotide repeat expansion by in situ hybridization. The disclosed method uses a sample of nucleated cells, a labeled trinucleotide repeat-specific probe and detection of the hybridized probe by a means whose sensitivity distinguishes between the signal from probes hybridized to an expanded repeat and the signal from probes hybridized to a non-expanded repeat.

Detection of trinucleotide repeats by in situ hybridization

Disclosed are methods for accurately determining the total emission intensity of a single fluorochrome, under imaging conditions, using a digital imaging fluorescence microscopy system. Also disclosed are methods for detecting and localizing probe-target molecule binding. The detection method has sufficient resolution and sensitivity to locate and detect a single target-bound probe.

Single molecule detection by in situ hybridization

Disclosed are methods for removing autofluorescence background from microscopic images or from flow cytometer measurements. The microscopic images can be fluorescent in situ hybridization images or images of living cells. The methods involve the steps of: (1) detecting fluorescence from a cell before 'uncaging' a caged fluorochrome label on a probe; (2) uncaging the fluorochrome; (3) detecting fluorescence from the cell; and (4) subtracting fluorescence detected before uncaging from fluorescence detected after uncaging. Also disclosed is a method for tracking the movement of a target molecule in a living cell. Also disclosed is a fluorochrome-uncaging flow cytometer. The fluorochrome-uncaging flow cytometer includes a first fluorescence excitation light beam, an uncaging light beam, a second fluorescence excitation light beam, an electronic data system, a first photodetector operably linked to the electronic data system, and a second photodetector operably linked to the electronic data system.

Caged fluorochrome-labeled probes and subtraction of autoflourescence

A method of nucleic acid hybridization in living cells is described, which is useful for detecting, quantitating and locating a specific nucleic acid in a cell or tissue, for selecting cells based on the expression or presence of a specific nucleic acid, and for monitoring the amount and location of a specific nucleic acid over time or under various inducing or inhibiting conditions.

Detection of hybridized oligonocleotide probes in living cells

Disclosed are the particular 3' noncoding nucleotide sequences (zipcode and zipcode elements) that cause .beta.-actin mRNA (and .beta.-actin protein synthesis) to be localized in specific regions of the cytoplasm. Also disclosed is the discovery that zipcode function can be fully inhibited by antisense oligonucleotides.

Cis-acting sequences for intracellular localization of RNA

Strand Displacement Amplification for amplification of nucleic acid target sequences in situ in cells in suspension, on slides or in tissues. Excellent specimen morphology is preserved, and either DNA targets, or RNA targets, or both may be selectively amplified. In situ amplification by SDA is compatible with immunochemical techniques, so that both amplification of target sequences and immunological staining can be performed on the same specimen.

Detection of nucleic acids in cells by strand displacement amplification

Improved methodologies for in-situ hybridization and detection of hybridized nucleic acid sequences in cell cultures and tissue sections are provided which offer an increase of speed, sensitivity, and simplicity unavailable in previously known techniques. The invention detects specific nucleic acids of interest, particularly RNA sequences, within cells and tissues utilizing DNA of a particular size as a probe to find those sequences which are held substantially in common between the cell or tissue and the probe. The cells are fixed preferably in paraformaldehyde and then hybridized using a hybridization fluid for not less than 10 minutes but not substantially more than 24 hours. A variety of identifying labels are attached to the probe which permit quick and rapid detection via measurement of radioactive isotope decay or by colorimetric detection of enzymatic reaction products. The invention is intended for use as a diagnostic kit in clinical/diagnostic laboratory testing facilities in that it permits a relatively unskilled person to accurately and reproducibly detect a few molecules of a specific nucleic acid of interest in-situ in 10 minutes.

Growing community of inventors

Shrewsbury, MA, United States of America

Robert H Singer