Nucleic acid hybridization has long been popularly used to detect nucleic acids that have been immobilized in some way. In some cases, the nucleic acid samples have been isolated from cells and immobilized by binding them to a solid support such as a nylon membrane. That can be done very simply by injecting individual nucleic acid samples onto the membrane so that they are bound at regular intervals (dot blot hybridization). In other cases, the isolated nucleic acids may have been size-fractionated by gel electrophoresis before being transferred to the solid support. Alternatively, the hybridization assay is designed to detect nucleic acids in situ within native structures such as cells or chromosomes.
Southern and northern blot hybridization
In Southern hybridization assays, a sample population of purified DNA is digested with one or more restriction endonucleases, generating fragments that are several hundred to many thousands of base pairs in length. The restriction fragments are separated according to size by agarose gel electrophoresis, denatured, and transferred to a nitrocellulose or nylon membrane. Labeled probes are hybridized to the membrane-bound target DNA and the positions of the labeled heteroduplexes are revealed by autoradiography (Figure1). Although this method has largely been superseded by PCR assays, it is still used in diagnostic assays that seek to identify large DNA changes (which are difficult to detect by PCR).
Fig1. Southern blot hybridization. A complex DNA sample is digested with restriction endonucleases. The resulting fragments are applied to an agarose gel and separated by size using electrophoresis. The gel is treated with alkali to denature the DNA fragments and is then placed against a nitrocellulose or nylon membrane. DNA will be transferred from the gel to the membrane, which is then soaked in a solution containing a radiolabeled, single-stranded DNA probe. After hybridization, the membrane is washed to remove excess probe and then dried. The membrane is then placed against an X-ray film and the position of the labeled probe will cause a latent image on the film that can be revealed by development of the autoradiograph as a hybridization band.
Northern blot hybridization is a variant of Southern blotting in which the samples contain undigested size-fractionated RNA instead of DNA. In the past, this method was regularly used to obtain information on which tissues genes were expressed in, and to identify tissue-specific isoforms. However, it has been superseded by RT-PCR and sequencing assays.
Chromosome in situ hybridization
In chromosome in situ hybridization, a suitable labeled DNA probe is hybridized against chromosomal DNA that has been denatured in situ. First, an air-dried, microscope-slide chromosome preparation is made, typically using metaphase or prometaphase chromosomes from peripheral blood lymphocytes or lymphoblastoid cell lines. RNA and protein are then removed from the sample by treatment with RNase and proteinase K, and the remaining chromosomal DNA is denatured by exposure to formamide. The denatured DNA is exposed to an added solution containing a labeled nucleic acid probe and over laid with a coverslip.
Depending on the particular technique, chromosome banding of the chromosomes can be arranged either before or after the hybridization step. As a result, the signal obtained after removal of excess probe can be correlated with the chromosome band pat tern in order to identify a map location for the DNA sequences recognized by the probe. Chromosome in situ hybridization has been revolutionized by the use of fluorescently labeled probes in fluorescence in situ hybridization (FISH) techniques; see Table 1 for cross-references to some figures containing example images.
Table1. POPULARLY USED HYBRIDIZATION ASSAYS
Tissue in situ hybridization
In tissue in situ hybridization, labeled probes are hybridized against RNA in tissue sections. Very thin tissue sections are cut, either from paraffin wax-embedded tissue blocks or from frozen tissue using a cryostat, and then mounted onto glass slides. A hybridization mix including the probe is applied to the section on the slide and covered with a glass coverslip. Single-stranded antisense RNA probes (riboprobes) are preferred.
The riboprobes can be labeled with a radioisotope such as 33P or 35S, and the hybridized probes visualized using autoradiographic procedures. The localization of the silver grains is often visualized using only dark-field microscopy. Here, direct light is not allowed to reach the objective; instead, the illuminating rays of light are directed from the side so that only scattered light enters the microscopic lenses and the signal appears as an illuminated object against a black background. However, better signal detection is possible using bright-field microscopy, in which the image is obtained by direct trans mission of light through the sample. Alternatively, probes are subjected to fluorescence labeling and detection is accomplished by fluorescence microscopy.
