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

• US 6,242,184 B1
• Filed: 02/25/1999
• Issued: 06/05/2001
• Est. Priority Date: 10/18/1988
• Status: Expired due to Term

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: Singer, Robert H., Johnson, Carol Villnave, Lawrence, Jeanne Bentley
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/259,099
• Time in Patent Office: 831 Days
• Field of Search: 435/6, 536/23.1, 536/25.3
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• 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

Abstract

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.

Claims

1. A method for detecting a target nucleic acid within cellular material by in-situ hybridization with an iterative effect whereby hybridized probe fragments line up along a strand of the target nucleic acid, thereby amplifying the signal from the target, comprising the steps of:

(a) fixing the cellular material with a fixative that retains and preserves the target nucleic acid and allows probe penetration of the cellular material;

(b) contacting the cellular material with a hybridization fluid comprising a non-homopolymeric probe consisting essentially of a multiplicity of non-radioactively labeled, heterogeneous nucleic acid fragments collectively complementary to a multiplicity of portions along said target nucleic acid, each fragment having from about 20 to 1,000 nucleotides, under conditions such that the probe hybridizes specifically to the target nucleic acid if present, said nucleic acid fragments selected or chemically synthesized so as to exclude substantially all nucleic acid fragments longer than 1,000 nucleotides;

(c) selecting a threshold of detection such that the cumulative effect of iterative signals are detectable while individual probe molecules are not detectable; and

(d) detecting the probe hybridized to said target nucleic acid if present.

2. The method of claim 1, wherein the target nucleic acid is an exogenous nucleic acid.

3. The method of claim 2, wherein the exogenous nucleic acid is an integrated viral nucleic acid.

4. The method of claim 3, wherein the integrated viral nucleic acid is a human immunodeficiency virus nucleic acid.

5. The method of claim 1 wherein the target nucleic acid is integrated into a pair of sister chromatids, with the target nucleic acid being detectable as a pair of signals.