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Licensing opportunity DI 05/39

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

Inventors: S. Wain-Hobson et al.

Description of invention: 

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 (Tp) 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 °C lower than Tp. 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.

 Specific advandages
 A quick and easy to perform method.

 Potential applications
 The identification of AT rich bacterial or microbial sequences in the presence of other bacteria. The identification of double or multiple infection by various strains if the same microbe that differ in their GC/AT content. The identification of alleles differing by a single GC->AT substitution as shown for the Ha-ras gene at codon 12. Hence it could be used to follow tumor regression following therapy of cancers involving this gene. This could be said of any such gene.
Patent Status:
 US patent application filed on December 30, 2005
 International patent application filed on December 28, 2006

 Recent pertinent publications:
 Interferon-inductible expression of APOBEC3 editing enzymes in human hepatocytes and inhibition of hepatitis B virus replication
Bonvin M, Achermann F, Greeve I, Stroka D, Keogh A, Inderbitzin D, Candinas D, Sommer
P, Wain-Hobson S, Vartanian JP, Greeve J.
Hepatology. 2006 Jun;3(6):1364-74. 
 Restriction of Foamy viruses bye APOBEC cytidine deaminases
Delebecque F, Suspene R, Calattini S, Casartelli N, Saib A, Froment A, Wain-Hobson S, Gessain A, Vartanian JP, Schwartz O.
J Virol. 2006 Jan;80(2):605-14. 
 Extensive editing of a small fraction of human T-cell leukemia virus type 1 genomes by four APOBEC3 cytidine deaminases
Mahieux R, Suspene R, Delebecque F, Henry M, Schwartz O, Wain-Hobson S, Vartanian JP.
J Gen Virol. 2005 Sep;86(Pt 9):2489-94.

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