Recovery of APOBEC3-edited human immunodeficiency virus G -> A hypermutants by differential DNA denaturation PCR

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 degreesC 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.