Differential DNA denaturation PCR using a temperature 1-3 degrees C lower than standard allowed selective amplification of AT-rich HIV-1 G-->A hypermutants and poliovirus variants.
Differential DNA denaturation PCR is a sensitive method for selectively amplifying AT-rich viral variants, such as HIV-1 G->A hypermutants.
Virus genomes from the same family may exhibit a wide range in their DNA GC content, whereas viral hypermutants differ substantially in GC content from their parental genomes. As AT-rich DNA melts at lower temperatures than GC-rich DNA, use of a lower denaturation temperature during PCR should allow differential amplification of AT-rich genomes or variants within a quasispecies. The latter situation has been explored explicitly in a two-step process by using a series of well-defined viral sequences differing in their AT content. Firstly, the lowest denaturation temperature (T(p)) that allowed amplification of the parental sequence was determined. Secondly, differential amplification of AT-rich viral variants was obtained by using a denaturation temperature 1-3 degrees C lower than T(p). Application of this sensitive method to two different viruses allowed us to identify human immunodeficiency virus type 1 G-->A hypermutants in a situation where none were expected and to amplify AT-rich variants selectively within a spectrum of poliovirus mutants.
Suspène et al. (2004) studied Human immunodeficiency virus type 1 and poliovirus. Differential DNA denaturation PCR vs. Standard PCR (denaturation temperature for parental sequence) was evaluated on Differential amplification of AT-rich viral variants. Differential DNA denaturation PCR using a temperature 1-3 degrees C lower than standard allowed selective amplification of AT-rich HIV-1 G-->A hypermutants and poliovirus variants.