Monitoring specific nucleic acid sequences:
The rate of establishing of double-stranded hybrids depends on the concentration of the two single-stranded species. That can be used to measure the concentration of either specific RNA or DNA sequences in a complex mixture. The first job is to prepare a single-stranded DNA probe for instance a DNA fragment which is complementary to the nucleic acid being assayed. This should be one strand of a DNA restriction fragment, a synthetic or cloned DNA oligonucleotide. It must be labeled in the order to be able to detect the formation of hybrids among it and the goal nucleic acid. While most labeling used to be through the incorporation of a radioisotope, nonradioactive chemical labels are now frequent used instead. For instance, a DNA probe can be labeled with digoxygenin, a steroid, through using digoxygenin-labeled dUTP during DNA synthesis. Hybrids holding the digoxygenin-labeled DNA probe can then be detected using antidigoxygenin antibody linked to a fluorescent dye. Irrespective of the technique of probe labeling the DNA probe is incubated with the nucleic acid sample (the 'target' DNA or RNA) and then nuclease is added to degrade any unhybridized single- stranded probe. The value of labeled probe remaining indicates the concentration of the target nucleic acid in the sample.
The hybridization conditions for example temperature and salt concentration can be varied so as to govern the kind of hybrids formed. The conditions should be arranged so which only perfectly matched hybrids are stable and therefore assayed conditions known as high stringency. In the other words, the conditions may be such which even poorly complementary hybrids are stable and will be detected low stringency. Therefore through varying the reaction conditions it is probably to detect and quantify only those goals sequences which are identical to the DNA alternatively or probe, to detect and quantify related sequences. The Hybridization of nucleic acid probes with genomic DNA, for instance, can be used to measure the copy number of particular DNA sequences in the genome. The Hybridization of a DNA probe with cellular RNA as the goal will indicate the concentration of the corresponding RNA transcript and therefore provides information about the stages of gene expression. Different of the methodology even permits determination of the transcriptional Start and Stop sites and the number and location of intron sequences in protein-coding genes.