GENE PROBES AND HYBRIDIZATION
June 1st, 2008 | by admin |We shall see in the following sections that what lies at the heart of gene analysis is the ability to identify a specific gene (or mRNA) in a complex mixture of all the DNA (or RNA) in a cell or tissue. This can only be done when one already has a cloned fragment of DNA from the gene of interest. Such fragments are usually obtained from gene libraries constructed from genomic DNA (described above) or cDNA (to be described below).
These DNA fragments can be almost any size, from a fraction of the size of the gene (a few hundred nucleotides) to the size of an entire gene (several thousand nucleotides). These cloned gene fragments are
called “probes” because they are used to probe native DNA or RNA for the gene of interest.
In order to be useful, a gene probe must contain a sufficient number of nucleotides so that it will recognize the sequences of its corresponding gene. Recognition occurs by a process called “nucleic acid hybridization” in which two pieces of DNA can align themselves (or “anneal”) by base-pairing. One can tag the probe DNA (e.g., using 32P-labeled nucleotides), split apart its two strands by heating (“denaturing”) and add it to the DNA mixture being studied, which has usually been immobilized by sticking it to an inert flat sheet. Under appropriate reannealing conditions, wherever the probe DNA finds a complementary sequence, it will base-pair with that sequence. All probes that have not specifically bound to their complementary DNA
targets can be washed away, and by exposing the flat sheet to x-ray film, the presence of the target DNA sequences can be revealed (see “Southern Blotting” below).
