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Direct measurement of T-cell receptor repertoire diversity with AmpliCot

Nature Methods volume 3, pages 895901 (2006) | Download Citation

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Abstract

Many studies require the measurement of nucleic acid sequence diversity. Here we describe a method, called AmpliCot, that measures the sequence diversity of PCR products on the basis of DNA hybridization kinetics, thereby avoiding the time, expense and biases associated with cloning and sequencing. SYBR Green dye is used to measure DNA hybridization kinetics in a homogeneous, automated fashion. PCR products are prepared in wholly double-stranded homoduplex form for a baseline measurement of DNA concentration. The DNA is melted and then reannealed under stringent conditions that allow only homoduplexes to form. The sequence diversity of a sample is proportional to the product of its concentration and the time required for it to anneal. After validating AmpliCot with a library of diverse sequences, we use it to measure the diversity of expressed rearrangements of the gene encoding the T-cell antigen receptor (TCR) β chain. AmpliCot measurements are in good agreement with previous estimates of murine TCR repertoire diversity that required extensive cloning and sequencing.

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Acknowledgements

We thank C. Stoddart, T. Liegler and K. Shokat for use of equipment; G. Chkhenkeli and N. Sapitan for assistance with mice; P. Harbury, M. Chamberlin, L. Hood, R. Britten and members of The J. David Gladstone Institutes for discussions; and J. Oksenberg, E. Delwart, C. Barker and laboratory colleagues for comments on the manuscript. The work was supported in part by grants from the US National Institutes of Health (including AI47062 and AI43864 to J.M.M.), and was conducted while P.B. was a Pfizer Postdoctoral Fellow. J.M.M. is a recipient of the Burroughs Wellcome Fund Clinical Scientist Award in Translational Research and the NIH Director's Pioneer Award Program, part of the NIH Roadmap for Medical Research (funded through grant number DPI OD00329).

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Affiliations

  1. Division of Experimental Medicine, Department of Medicine, San Francisco General Hospital, University of California, San Francisco, California, 94110, USA.

    • Paul D Baum
    •  & Joseph M McCune

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Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Joseph M McCune.

Supplementary information

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    Supplementary Fig. 1

    T-cell receptor mRNA expression.

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    Supplementary Fig. 2

    Interassay variability.

  3. 3.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth949

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