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    J Gen Virol. 2005 Jan;86(Pt 1):125-9.

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

    Source

    Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.

    Abstract

    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.

    PMID:
    15604439
    [PubMed - indexed for MEDLINE]
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