Several nucleotide probes are used in in situ hybridization. Oligonucleotide probes can be as small as 20 to 40 base pairs. Single-or double-stranded DNA probes are much longer and can contain as many as 1,000 base pairs. For specific localization of mRNA, complementary RNA probes are used. These probes are labeled with radioactive isotopes (e.g., 32P, 35S, 3H), a specifically modified nucleotide (digoxigenin), or biotin (a commonly used covalent multipurpose label). Radioactive probes can be detected and visualized by autoradiography. Digoxigenin and biotin are detected by immunocytochemical and cytochemical methods, respectively.
The strength of the bonds between the probe and the complementary sequence depends on the type of nucleic acid in the two strands. The strongest bond is formed between a DNA probe and a complementary DNA strand and the weakest between an RNA probe and a complementary RNA strand. If a tissue specimen is expected to contain a minute amount of mRNA or a viral transcript, then polymerase chain reaction (PCR) amplification for DNA or reverse transcriptase-PCR (RT-PCR) for RNA can be used. The amplified transcripts obtained during these procedures are usually detected using labeled complementary nucleotide probes in standard in situ hybridization techniques.
Recently, fluorescent dyes have been combined with nucleotide probes, making it possible to visualize multiple probes at the same time (Fig. 1.7). This technique, called
FIGURE 1.7 • Example of the FISH technique used in a prenatal screening test. Interphase nuclei of cells obtained from amniotic fluid specimens were hybridized with two specific DNA probes. The orange probe (LSI 21) is locus specific for chromosome 21, and the green probe (LSI 13) is locus specific for chromosome 13. The right nucleus is from a normal amniotic fluid specimen and exhibits two green and two orange signals, which indicates two copies of chromosomes 13 and 21, respectively. The nucleus on the left has three orange signals, which indicate trisomy 21 (Down syndrome). DNA has been counterstained with a nonspecific blue stain (DAPI stain) to make the nucleus visible. 1,250. (Courtesy of Dr. Robert B. Jenkins.)