Stringency


Stringency is the extent to which hybridisation can occur between nucleic acids with mismatched sequences. High-stringency conditions require absolute complementarity between the molecules, while low-stringency conditions permit hybridisation where there are some mismatched bases. Nucleic acids that are perfectly matched, under high-stringency conditions, are said to be homologous; nucleic acids that hybridise despite some base mismatches, under low-stringency conditions, are said to be heterologous.

Typically, high-stringency conditions are achieved either by reducing NaCl concentration or increasing temperature, close to the melt temperature (Tm) of the molecules involved. Lower-stringency conditions are achieved by increasing NaCl concentration or decreasing temperature considerably below the Tm of the molecules. The level of stringency can be varied by adjusting temperature, salt concentration or formamide concentration; this is important when, for example, searching for a single-base mutation in a DNA sequence.

Increased formamide concentration reduces the melt temperature of hybrids (it does this by disrupting hydrogen bonds). Formamide is a useful tool in hybridisation because it enables hybrids to be formed at lower temperatures: this is crucial in Northern blotting, for example, where DNA-RNA hybrids need to be formed at a temperature that will not degrade the RNA.

The level of stringency used in hybridisation depends on the purpose of hybridisation. For example, when probing for a specific gene sequence on a microarray chip, a very high level of specificity is required and thus high-stringency conditions will apply. However, when dealing with organismal comparisons, for example comparing the gene sequence for the same enzyme in a human and a chimpanzee, some sequence mismatch is expected and thus lower-stringency conditions will apply.

Standard saline citrate is a solution of 0.15M NaCl and 0.015M sodium citrate. This is usually used at 5-6x for hybridisation under low-stringency conditions (i.e. for heterologous probing) or 0.1x for high-stringency conditions (i.e. for homologous probing).