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 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
Rodolphe
R
Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Henry
Michel
M
Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Guillot
Sophie
S
Unité de Recherche Prévention et Thérapie moléculaires des Maladies humaines, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Wain-Hobson
Simon
S
Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
Vartanian
Jean-Pierre
JP
Unité de Rétrovirologie Moléculaire, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15, France.
D001483:Base Sequence; D002460:Cell Line; D043525:Cytosine Deaminase / Q000235:genetics*; D004279:DNA, Viral / Q000378:metabolism*; D005814:Genes, Viral; D015699:HIV Envelope Protein gp120 / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017393:RNA Editing; D012367:RNA, Viral / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D004279:DNA, Viral; D015699:HIV Envelope Protein gp120; C059761:HIV envelope protein gp120 (135-148); D010446:Peptide Fragments; D012367:RNA, Viral; C492906:APOBEC3F protein, human; D043525:Cytosine Deaminase