Multiarm high-throughput integration site detection: Limitations of LAM-PCR technology and optimization for clonal analysis

Michael A. Harkey, Rajinder Kaul, Michael A. Jacobs, Peter Kurre, Don Bovee, Ruth Levy, C. Anthony Blau

Research output: Contribution to journalArticle

31 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)381-392
Number of pages12
JournalStem Cells and Development
Volume16
Issue number3
DOIs
StatePublished - Jun 2007

ASJC Scopus subject areas

  • Hematology
  • Developmental Biology
  • Cell Biology

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