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Direct measurement of T-cell receptor repertoire diversity with AmpliCot

Nature Methods volume 3, pages 895–901 (2006)Cite this article

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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Figure 1: Overview of AmpliCot.
Figure 2: Measurement of sequence diversity with hybridization kinetics.
Figure 3: Monitoring hybridization stringency.
Figure 4: Titration of input diversity.
Figure 5: Validation of AmpliCot with samples previously studied with sequencing methods.
Figure 6: Effect of expanded lymphocyte clones on AmpliCot measurements of TCR repertoire.

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References

  1. Britten, R.J. & Kohne, D.E. Repeated sequences in DNA. Science 161, 529–540 (1968).

    Article  CAS  Google Scholar 

  2. Casrouge, A. et al. Size estimate of the αβ TCR repertoire of naive mouse splenocytes. J. Immunol. 164, 5782–5787 (2000).

    Article  CAS  Google Scholar 

  3. Thompson, J.R., Marcelino, L.A. & Polz, M.F. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by 'reconditioning PCR'. Nucleic Acids Res. 30, 2083–2088 (2002).

    Article  CAS  Google Scholar 

  4. Pannetier, C. et al. The sizes of the CDR3 hypervariable regions of the murine T-cell receptor β chains vary as a function of the recombined germ-line segments. Proc. Natl. Acad. Sci. USA 90, 4319–4323 (1993).

    Article  CAS  Google Scholar 

  5. Pannetier, C., Even, J. & Kourilsky, P. T-cell repertoire diversity and clonal expansions in normal and clinical samples. Immunol. Today 16, 176–181 (1995).

    Article  CAS  Google Scholar 

  6. Arstila, T.P. et al. A direct estimate of the human αβ T cell receptor diversity. Science 286, 958–961 (1999).

    Article  CAS  Google Scholar 

  7. Garboczi, D.N. et al. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384, 134–141 (1996).

    Article  CAS  Google Scholar 

  8. Garcia, K.C. et al. An αβ T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 274, 209–219 (1996).

    Article  CAS  Google Scholar 

  9. Suzuki, M.T. & Giovannoni, S.J. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625–630 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Mathieu-Daude, F., Welsh, J., Vogt, T. & McClelland, M. DNA rehybridization during PCR: the 'Cot effect' and its consequences. Nucleic Acids Res. 24, 2080–2086 (1996).

    Article  CAS  Google Scholar 

  11. Ririe, K.M., Rasmussen, R.P. & Wittwer, C.T. Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154–160 (1997).

    Article  CAS  Google Scholar 

  12. Shen, D.F., Doukhan, L., Kalams, S. & Delwart, E. High-resolution analysis of T-cell receptor β-chain repertoires using DNA heteroduplex tracking. J. Immunol. Methods 215, 113–121 (1998).

    Article  CAS  Google Scholar 

  13. Yguerabide, J. & Ceballos, A. Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator. Anal. Biochem. 228, 208–220 (1995).

    Article  CAS  Google Scholar 

  14. Britten, R.J. & Davidson, E.H. Hybridization strategy. In Nucleic Acid Hybridisation: a Practical Approach (eds. Hames, B.D. & Higgins, S.J.) 3–15 (IRL, Oxford, 1985).

    Google Scholar 

  15. Gilfillan, S., Dierich, A., Lemeur, M., Benoist, C. & Mathis, D. Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 261, 1175–1178 (1993).

    Article  CAS  Google Scholar 

  16. Cabaniols, J.P., Fazilleau, N., Casrouge, A., Kourilsky, P. & Kanellopoulos, J.M. Most α/β T cell receptor diversity is due to terminal deoxynucleotidyl transferase. J. Exp. Med. 194, 1385–1390 (2001).

    Article  CAS  Google Scholar 

  17. Arstila, T.P. et al. Diversity of human αβ T cell receptors. Science 288, 1135 (2000).

    Article  CAS  Google Scholar 

  18. Soen, Y., Chen, D.S., Kraft, D.L., Davis, M.M. & Brown, P.O. Detection and characterization of cellular immune responses using peptide-MHC microarrays. PLoS Biol. 1, 429–438 (2003).

    Article  CAS  Google Scholar 

  19. Wagner, U.G., Koetz, K., Weyand, C.M. & Goronzy, J.J. Perturbation of the T cell repertoire in rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 95, 14447–14452 (1998).

    Article  CAS  Google Scholar 

  20. Naylor, K. et al. The influence of age on T cell generation and TCR diversity. J. Immunol. 174, 7446–7452 (2005).

    Article  CAS  Google Scholar 

  21. Ogle, B.M. et al. Direct measurement of lymphocyte receptor diversity. Nucleic Acids Res. 31, e139 (2003).

    Article  Google Scholar 

  22. Cohn, M. & Langman, R.E. The immune system: a look from a distance. Front. Biosci. 1, 318–323 (1996).

    Article  Google Scholar 

  23. Halpin, D.R. & Harbury, P.B. DNA display I. Sequence-encoded routing of DNA populations. PLoS Biol. 2, E173 (2004).

    Article  Google Scholar 

  24. Ellington, A.D. & Szostak, J.W. In vitro selection of RNA molecules that bind specific ligands. Nature 346, 818–822 (1990).

    Article  CAS  Google Scholar 

  25. Adleman, L.M. Molecular computation of solutions to combinatorial problems. Science 266, 1021–1024 (1994).

    Article  CAS  Google Scholar 

  26. Glusman, G. et al. Comparative genomics of the human and mouse T cell receptor loci. Immunity 15, 337–349 (2001).

    Article  CAS  Google Scholar 

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

    Paul D Baum & Joseph M McCune

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  1. Paul D Baum
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Correspondence to Joseph M McCune.

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Supplementary information

Supplementary Fig. 1

T-cell receptor mRNA expression. (PDF 357 kb)

Supplementary Fig. 2

Interassay variability. (PDF 226 kb)

Supplementary Methods (PDF 55 kb)

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Baum, P., McCune, J. Direct measurement of T-cell receptor repertoire diversity with AmpliCot. Nat Methods 3, 895–901 (2006). https://doi.org/10.1038/nmeth949

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