Flanking-sequence exponential anchored-polymerase chain reaction amplification: a sensitive and highly specific method for detecting retroviral integrant-host-junction sequences.
BACKGROUND
Retroviral vectors are regularly used to transduce stem cells and their derivatives for experimental and therapeutic purposes. Because these vectors integrate semi-randomly into the cellular genome, analysis of integranated retroviral DNA/host cell DNA junctions (IHJ) facilitates clonality studies of engrafted cells, allowing their differentiation, survival and fate to be tracked. In the case of any adverse events, IHJ analysis can allow the identification of potentially oncogenic integration sites. At present, most measures to assess IHJ are complex, insensitive and may be subject to IHJ selection bias inherent to the technology used.


METHODS
We have developed and validated a simple but effective technique for generating libraries of IHJ, which we term flanking-sequence exponential anchored-polymerase chain reaction (FLEA-PCR). Flanking-sequence random anchoring is used as an alternative to restriction enzyme digestion and cassette ligation to allow consistent detection of IHJ and decrease bias.


RESULTS
Individual clones from plasmid libraries can be sequenced and assembled using custom-written software, and FLEA-PCR smears can be analyzed by capillary electrophoresis after digestion with restriction enzymes.


CONCLUSIONS
This approach can readily analyze complex mixtures of IHJ, allowing localization of these sequences to their genomic sites. This approach should simplify analysis of retroviral integration.
Pule
M A
MA
Center for Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA. mpule@ucl.ac.uk
Rousseau
A
A
Vera
J
J
Heslop
H E
HE
Brenner
M K
MK
Vanin
E F
EF
D002469:Cell Separation; D004247:DNA / Q000032:analysis*; D045566:DNA, Cruciform / Q000032:analysis*; D004279:DNA, Viral / Q000032:analysis*; D005434:Flow Cytometry; D005822:Genetic Vectors; D006367:HeLa Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D007963:Leukocytes, Mononuclear; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012190:Retroviridae / Q000235:genetics*; D020079:Terminal Repeat Sequences / Q000235:genetics; D014161:Transduction, Genetic; D016662:Virus Integration / Q000235:genetics*
D045566:DNA, Cruciform; D004279:DNA, Viral; D004247:DNA