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

Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Research Abstract Details 

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Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Abstract Text:

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

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.

Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Publishing Authors By Initials

MA Harkey,R Kaul,MA Jacobs,P Kurre,D Bovee,R Levy,CA Blau,MA Harkey,R Kaul,MA Jacobs,P Kurre,D Bovee,R Levy,CA Blau,

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Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis. Journal Published:

PUBLICATION TYPE: Research Support, N.I.H., Extr

Journal: Stem cells and development

VOLUME: 16

Page Numbers: 381-92

Journal Abbreviation: Stem Cells Dev.

ISSN: 1547-3287

DAY: 5

MONTH: Jun

YEAR: 2007

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

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LANGUAGE: eng

NlmUniqueID: 101197107

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Grant and Affiliation Information for Multiarm high-throughput integration site detection: limitations of LAM-PCR technology and optimization for clonal analysis.

AFFILIATION: Department of Transplantation Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA. mharkey@fhcrc.org

Country: United States

AGENCY: United States NIDDK

GRANT: R01 DK61844-01

ACRONYM: DK

MEDLINETA: Stem Cells Dev

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