Methods for assessing genetic and phenotypic markers by simultaneous multicolor visualization of chromogenic dyes using brightfield microscopy and spectral imaging

• US 6,294,331 B1
• Filed: 08/07/1998
• Issued: 09/25/2001
• Est. Priority Date: 08/08/1997
• Status: Expired due to Term

• Current Assignee: National Institutes of Health (NIH) (Government of the United States of America)
• Original Assignee: United States Department Of Health And Human Services (Government of the United States of America)
• Inventors: Ried, Thomas, MacVille, Merryn V. E., Hopman, Anton H. N.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/130,078
• Time in Patent Office: 1,145 Days
• Field of Search: 435/6, 435/7.1, 435/7.92, 536/24.3, 359/368
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

The present invention is directed to an improved method for detecting a genetic marker in a biological sample comprising contacting the biological sample with a nucleic acid probe linked to a detectable moiety, whereby the detectable moiety can be detected by the presence of a chromogenic dye associated with the detectable moiety, obtaining a spectral image of the biological sample using brightfield microscopy, and detecting the presence of the chromogenic dye, thereby detecting the genetic marker in the biological sample. The present invention also provides an improved method for detecting a phenotypic marker in a biological sample comprising contacting the biological sample with a compound comprising a detectable moiety, whereby the compound associates with the phenotypic marker and whereby the detectable moiety can be detected by the presence of a chromogenic dye associated with the detectable moiety, obtaining a spectral image of the biological sample using brightfield microscopy, and detecting the presence of the chromogenic dye, thereby detecting the phenotypic marker in the biological sample.

Claims

1. A method for detecting a genetic marker in a biological sample comprising:

a) contacting the biological sample with a nucleic acid probe linked to a detectable moiety, whereby the detectable moiety can be detected by the presence of a chromogenic dye associated with the detectable moiety;

b) obtaining a spectral image of the biological sample using brightfield microscopy; and

c) detecting the presence of the chromogenic dye, thereby detecting the genetic marker in the biological sample.

2. The method of claim 1, wherein the biological sample is contacted with nucleic acid probes having at least two different specificities.

3. The method of claim 1, wherein the biological sample is attached to a substrate.

4. The method of claim 1, wherein step (a) further comprises contacting the biological sample with a cytological stain.

5. The method of claim 1, wherein step (c) further comprises distinguishing the cytological stain from the detectable moiety.

6. The method of claim 1, wherein the genetic marker is selected from the group consisting of a centromere, a telomere, a general genetic loci, a specific genetic loci, a chromosome band, a chromosome-specific loci, a chromosome fragment, and a whole chromosome.

7. The method of claim 1, wherein the detectable moiety comprises a hapten.