title: Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis.
abstract: Retroviral integration provides a unique and heritable genomic tag for a target cell and its progeny, enabling studies of clonal composition and repopulation kinetics after gene transfer into hematopoietic stem cells. The clonal tracking method, linear amplification-mediated polymerase chain reaction (LAM-PCR) is widely employed to follow the hematopoietic output of retrovirally marked stem cells. Here we examine the capabilities and limitations of conventional LAM-PCR to track individual clones in a complex multiclonal mix. Using artificial mixtures of retrovirally marked, single-cell-derived clones, we demonstrate that LAM-PCR fails to detect 30-40% of the clones, even after exhaustive analysis. Furthermore, the relative abundance of specific clones within a mix is not accurately represented, deviating by as much as 60-fold from their true abundance. We describe an optimized, multiarm, high-throughput modification of LAM-PCR that improves the global detection capacity to greater than 90% with exhaustive sampling, facilitates accurate estimates of the total pool size from smaller samplings, and provides a rapid, cost-effective approach to the generation of large insertion-site data bases required for evaluation of vector integration preferences. The inability to estimate the abundance of individual clones within mixtures remains a serious limitation. Thus, although LAM-PCR is a powerful tool for identification of integration sites and for estimations of clonal complexity, it fails to provide the semiquantitative information necessary for direct, reliable tracking of individual clones in a chimeric background.
authors.affiliation: Department of Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. mharkey@fhcrc.org
authors.forename: Michael A
authors.initials: MA
authors.lastname: Harkey
authors.forename: Rajinder
authors.initials: R
authors.lastname: Kaul
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jacobs
authors.forename: Peter
authors.initials: P
authors.lastname: Kurre
authors.forename: Don
authors.initials: D
authors.lastname: Bovee
authors.forename: Ruth
authors.initials: R
authors.lastname: Levy
authors.forename: C Anthony
authors.initials: CA
authors.lastname: Blau
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002999:Clone Cells / Q000166:cytology / Q000502:physiology*; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D015203:Reproducibility of Results; D012190:Retroviridae / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D049452:Green Fluorescent Proteins