Method for simultaneous detection of multiple fluorophores for in situ hybridization and multicolor chromosome painting and banding

• US 6,066,459 A
• Filed: 06/19/1998
• Issued: 05/23/2000
• Est. Priority Date: 08/18/1993
• Status: Expired due to Fees

• Current Assignee: Applied Spectral Imaging Ltd.
• Original Assignee: Applied Spectral Imaging Ltd.
• Inventors: Garini, Yuval, Soenksen, Dirk G., Cabib, Dario, Ried, Thomas, Buckwald, Robert A.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/100,104
• Time in Patent Office: 704 Days
• Field of Search: 435/6, 536/24.3, 536/24.31
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G01J 2003/2866 Markers; Calibrating of scan
• CPC Class Codes: G01J 3/02 Details
• CPC Class Codes: G01J 3/12 Generating the spectrum; Monochromators
• CPC Class Codes: G01J 3/1256 using acousto-optic tunable filter; acousto-optic elements or systems G02F1/11, G02F1/33
• CPC Class Codes: G01J 3/26 using multiple reflection, e.g. Fabry-Perot interferometer, variable interference filters
• CPC Class Codes: G01J 3/2823 Imaging spectrometer
• CPC Class Codes: G01J 3/4406 Fluorescence spectrometry
• CPC Class Codes: G01J 3/453 by correlation of the amplitudes
• CPC Class Codes: G01N 2021/6417 Spectrofluorimetric devices
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/6458 Fluorescence microscopy fluorescence microscopes per se G02B21/0076 and G02B21/16
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/582 : with fluorescent label
• CPC Class Codes: G06V 10/88 : Image or video recognition using optical means, e.g. reference filters, holographic masks, frequency domain filters or spatial domain filters
• CPC Class Codes: G06V 20/69 : Microscopic objects, e.g. biological cells or cellular parts

Abstract

A spectral imaging method for simultaneous detection of multiple fluorophores aimed at detecting and analyzing fluorescent in situ hybridizations employing numerous chromosome paints and/or loci specific probes each labeled with a different fluorophore or a combination of fluorophores for color karyotyping, and at multicolor chromosome banding, wherein each chromosome acquires a specifying banding pattern, which pattern is established using groups of chromosome fragments labeled with various fluorophore or combinations of fluorophores.

Claims

1. A method of hybridization based multicolor chromosome banding comprising the steps of:

(a) providing a metaphase chromosome spread of a first species and chromosome paints of a second species, at least some of said chromosome paints being labeled with a different fluorophore or combination of fluorophores;

(b) hybridizing said chromosome paints with said metaphase chromosome spread;

(c) using a fluorescence microscope being optically coupled to a multiband collection device for collecting spectral data from said chromosome spread; and

(d) based on said collected data calculating a color image in which pixels associated with each of said different fluorophores or combination of fluorophores are attributed distinctive colors, thereby a banded color image of said metaphase chromosome spread is obtainable;

wherein said method is for an application selected from the group consisting of providing a color banding karyotype of embryonic cells, providing a color banding karotype of white blood cells, providing a color banding karyotype of malignant cells, providing a color banding karyotype of cells examined for malignancy, detecting the stage of a malignancy, following the efficiency of an anti-cancer treatment and detecting chromosomal aberrations following exposure to mitogenic agents.

2. A method as in claim 1, where in said fluorescence microscope is supplemented with a filter selected from the group consisting of a double band dichroic filter and a triple band dichroic filter.

3. A method as in claim 1, wherein said chromosome paints are labeled using a strategy selected from the group consisting of combinatorial labeling and combinatorial hybridization.

4. A method as in claim 1, wherein said chromosome paints are labeled by interspersed repetitive sequence--PCR.

5. A method as in claim 1, wherein said fluorophores are conjugated to a nucleotide or a nucleotide analog.

6. A method as in claim 1, wherein said first and second species are each independently selected from the group consisting of, human, mouse and monkey.

7. A method as in claim 1, wherein said embryonic cells are selected from the group consisting of chorionic villi cells and embryonic cells isolated from the peripheral blood of a pregnant woman.

8. A method as in claim 7, wherein said method is for detecting a trisomy of a genetic material selected from the group consisting of human chromosome 21, human chromosomal band 21q22, a fragment of human chromosomal band 21q22, human chromosome 18, a fragment of human chromosome 18, human chromosome 13 and a fragment of human chromosome 13.

9. A method as in claim 7, wherein said providing said color banding karyotype of said cells examined for malignancy, is for obtaining a color translocation map.

10. A method as in claim 1, wherein said detection of the stage of said malignancy is for selecting a treatment for said malignancy.

11. A method as in claim 1, wherein said metaphase chromosome spread is stained with a conventional chromosome banding dye, the method further comprising the step of obtaining a gray level banding image of said chromosomes.

12. A method as in claim 11, wherein said conventional chromosome banding dye is DAPI.

13. A method as in claim 1, wherein said multiband collection device is an imaging spectrometer.