DNA probes are created in several ways. A fragment of the gene to be analyzed can be cloned on a bacterial plas-mid and then isolated by restriction enzyme digestion and gel purification. The fragment, after labeling (see below) and denaturation, can then be used in Southern or Northern blot procedures.
Other sources of DNA probes include the isolation of a sequence of interest from viral genomes and in vitro organic synthesis of a piece of nucleic acid that has a particular sequence. The latter is used only for short, oligomeric probes. Probes can also be synthesized using the polymerase chain reaction (PCR) (see Chapter 7) to generate large amounts of specific DNA sequences.
The length of the probe will, in part, determine the specificity of the hybridization reaction. Probe lengths range from tens to thousands of base pairs. In analysis of the entire genome in a Southern blot, longer probes are more specific for a DNA region because they must match a longer sequence on the target. Shorter probes are not usually used in Southern blots because short sequences are more likely to be found in multiple locations in the genome, resulting in high background binding to sequences not related to the target region of interest. Short probes are more appropriate for mutational analysis as they are sensitive to single base mismatches (see Chapter 8). The probe is constructed so that it has a complementary sequence to the targeted gene. In order to bind to the probe then, the target nucleic acid has to contain the sequence of interest. There are typically fewer copies of a specific sequence in the genome, and therefore only a few bands will be apparent after detection.
Properly prepared and stored DNA probes are relatively stable and easy to manufacture. Double-stranded DNA probes must be denatured before use. This is usually accomplished by heating the probe (e.g., 95 C, 10-15 min) in hybridization solution or treating with 50% formamide/2X SSC at a lower temperature for a shorter time (e.g., 75C, 5-6 min)
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