3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple, simultaneous RNA detection

We have used fluorescein-, digoxigenin- and biotin-(di)deoxyXTPs and terminal deoxynucleotidyl transferase for small scale labeling of synthetic oligonucleotide probes and here we show the applicability of such probes for the in situ detection of multiple RNA sequences. The enzymatic 3'-end-labeling methods proved to be good alternatives for the chemical fluorochrome and hapten labeling of 5'-end alkylamino-derivatized oligonucleotides. By combining 3'-end fluorescein-, biotin-, and digoxigenin-labeled oligonucleotides, double and triple hybridizations are feasible. For example, we demonstrated simultaneously mRNAs coding for caudodorsal cell hormone, a molluscan insulin-related peptide, and 28 S ribosomal RNA in cryostat sections of the cerebral ganglia of the pond snail Lymnaea stagnalis.     

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.     

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

In situ hybridization: general infectivity assay for retroviruses.

We have devised a general infectivity assay for retroviruses. A virus-specific [32P]DNA probe is hybridized in situ to a monolayer culture, and foci of infected cells in the monolayer are detected by exposure of the hybridized culture to X-ray films. The method is quantitative, in that it gives the same titer for Moloney murine leukemia virus as does the standard UV-XC test. The specificity of the assay is indicated by the fact that murine leukemia virus and baboon endogenous virus do not cross hybridize under the conditions used. The assay is completed within 1 to 3 weeks and should be broadly applicable for retroviruses which replicate without altering cellular morphology: its use is demonstrated with mouse mammary tumor virus and the helper virus of the reticuloendotheliosis complex.     

Effects of various fluorochromes and competition between labeled oligonucleotides on their hybridization to oligonucleotides immobilized on biological microchips

Some technical tips are described on how to improve the discrimination between perfect and imperfect duplexes formed by hybridization of fluorescently labeled oligonucleotides to biological microchips. Model experiments were performed to assess the precision of the method. Effects of labeling on the efficiency of hybridization and some properties of competitive hybridization were studied using short synthetic oligonucleotides and three most popular fluorochromes as examples.     

Synthesis and purification of horseradish peroxidase-labeled oligonucleotides for tyramide-based fluorescence in situ hybridization

A method is presented to conjugate horseradish peroxidase (HRP) to oligodeoxynucleotides for fluorescence in situ hybridization assays employing tyramide signal amplification (TSA). HRP is covalently bound to the oligonucleotide by thiol ether linkage and purified by high-performance liquid chromatography. With TSA detection, a single HRP-labeled oligonucleotide probe is sufficient for in situ detection of clustered DNA repeat sequences with a degree of repetition between 20 and 50. Accepted: 6 December 1999     

In situ hybridization of nucleic acids

Hybridization of radioactive nucleic acid sequences can be carried out with DNA in cytological preparations. By autoradiography distinct DNA sequences can then be localized in eukaryotic chromosomes.