Tm Modulation

Any technique that involves hybridization of multiple sequences simultaneously, as in DNA chip and reverse hybridization technologies, is subject to inaccuracies due to differences in GC content. Sequences with high GC content may contain mismatches and still hybridize, whereas a low GC content probe may match perfectly and yet disassociate from the target, leading to false positives and negatives, respectively.

An elegant way of circumventing this problem would be to use a modified base that normalized the stability of the GC and AT base pairs. The N4-ethyl analogue (N4-Et-dC) hybridizes specifically to natural dG but the stability of the base pair is reduced to about the level of an AT base pair. In a series of probes whose GC content ranged from 0 to 100%, the range in Tm values when N4-Et-dC was used was only 7 °C; when dC was used, that range was 39 °C.