In-situ hybridization to detect nucleic acid sequences in morphologically intact cells

摘要

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.