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Immunol. Today
, vol.13
, pp. 306
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Alt, F.W.1
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2
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0029559690
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F. W. Alt et al., Immunol. Today 13, 306 (1992); B. Arden, S. P. Clark, D. Kabelitz, T. W. Mak, Immunogenetics 42, 501 (1995); L. Rowen, B. F. Koop, L. Hood, Science 272, 1755 (1996).
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Immunogenetics
, vol.42
, pp. 501
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Arden, B.1
Clark, S.P.2
Kabelitz, D.3
Mak, T.W.4
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3
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0030017526
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F. W. Alt et al., Immunol. Today 13, 306 (1992); B. Arden, S. P. Clark, D. Kabelitz, T. W. Mak, Immunogenetics 42, 501 (1995); L. Rowen, B. F. Koop, L. Hood, Science 272, 1755 (1996).
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, vol.272
, pp. 1755
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Rowen, L.1
Koop, B.F.2
Hood, L.3
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6
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0027251463
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Our approach was based on the immunoscope methodology [C. Pannetier et al., Proc. Natl. Acad. Sci. U.S.A. 90, 4319 (1993); C. Pannetier, J. Even, P. Kourilsky, Immunol. Today 16, 176 (1995)]. Complementary DNA is amplified with primers in the TCR V and C segments, followed by a run-off reaction with a nested fluorescent C gene- or J gene-specific primer. The products are analyzed with an Applied Biosystems PE373 automatic sequencer. The bands are expressed in graphic form as peaks, the area of each peak corresponding to the intensity of the band. A normal, polyclonal repertoire produces a Gaussian distribution of different CDR3 lengths, centering around 9 to 10 amino acids. An immune response can be detected as a deviation from the Gaussian distribution, the proliferating cells causing some peaks to expand in relation to others.
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(1993)
Proc. Natl. Acad. Sci. U.S.A.
, vol.90
, pp. 4319
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Pannetier, C.1
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7
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0028908698
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Our approach was based on the immunoscope methodology [C. Pannetier et al., Proc. Natl. Acad. Sci. U.S.A. 90, 4319 (1993); C. Pannetier, J. Even, P. Kourilsky, Immunol. Today 16, 176 (1995)]. Complementary DNA is amplified with primers in the TCR V and C segments, followed by a run-off reaction with a nested fluorescent C gene- or J gene-specific primer. The products are analyzed with an Applied Biosystems PE373 automatic sequencer. The bands are expressed in graphic form as peaks, the area of each peak corresponding to the intensity of the band. A normal, polyclonal repertoire produces a Gaussian distribution of different CDR3 lengths, centering around 9 to 10 amino acids. An immune response can be detected as a deviation from the Gaussian distribution, the proliferating cells causing some peaks to expand in relation to others.
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(1995)
Immunol. Today
, vol.16
, pp. 176
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Pannetier, C.1
Even, J.2
Kourilsky, P.3
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8
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0345679090
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note
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The CDR3 sequences were extracted from the raw sequence data with software developed by E. Beaudoing and others at Information Genetique et Structurale, CNRS, Marseille, France. Because of the infidelity of the Taq DNA polymerase, we excluded from the analysis sequences that were likely to represent polymerase chain reaction (PCR) mistakes, defined as sequences differing from another sequence by a single nucleotide. Analysis of conserved areas in the V gene showed that the actual rate of PCR mistakes was 1.6 per 1000 base pairs, that is, a mistake in 5% of a set of 10-amino acid segments. Our criterium resulted in the exclusion of 6% of the CDR3 sequences, a rate slightly in excess of the predicted amount. Thus, the data reported may be a slight underestimate.
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9
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0345679089
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note
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The relative intensity of a band was calculated by comparing it to the combined intensity of all bands in the V-J rearrangement. To test the correlation between the intensity of the band and the sequence diversity within it, we isolated two bands from the same V-J rearrangement. In bands with relative intensities of 2.5 and 13.5%, we found 5 and 34 different sequences, respectively. Thus, the differences in the intensity and in the diversity were 5.4- and 6.8-fold, respectively. In another experiment, peaks of 4 and 11% contained 45 and 150 different sequences, respectively (15), that is, a 2.8-fold difference in intensity and a 3.3-fold difference in diversity.
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10
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0026571274
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L. Ferradini and J. Even, unpublished material
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β genes was based on the sequencing of 427 random β chains [from W. M. C. Rosenberg, P. A. H. Moss, J. I. Bell, Eur. J. Immunol. 22, 541 (1992); L. Ferradini and J. Even, unpublished material]. These estimates correlated well with values we obtained by measuring the J-gene expression by semiquantitative PCR and also with other published estimates [for example, M. A. Hall and J. S. Lanchbury, Hum. Immunol. 43, 207 (1995); T. Nanki, H. Kohsaka, N. Miyasaka, J. Immunol. 161, 228 (1998)].
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Eur. J. Immunol.
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Rosenberg, W.M.C.1
Moss, P.A.H.2
Bell, J.I.3
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11
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0029080819
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β genes was based on the sequencing of 427 random β chains [from W. M. C. Rosenberg, P. A. H. Moss, J. I. Bell, Eur. J. Immunol. 22, 541 (1992); L. Ferradini and J. Even, unpublished material]. These estimates correlated well with values we obtained by measuring the J- gene expression by semiquantitative PCR and also with other published estimates [for example, M. A. Hall and J. S. Lanchbury, Hum. Immunol. 43, 207 (1995); T. Nanki, H. Kohsaka, N. Miyasaka, J. Immunol. 161, 228 (1998)].
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(1995)
Hum. Immunol.
, vol.43
, pp. 207
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Hall, M.A.1
Lanchbury, J.S.2
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12
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0031831004
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β genes was based on the sequencing of 427 random β chains [from W. M. C. Rosenberg, P. A. H. Moss, J. I. Bell, Eur. J. Immunol. 22, 541 (1992); L. Ferradini and J. Even, unpublished material]. These estimates correlated well with values we obtained by measuring the J- gene expression by semiquantitative PCR and also with other published estimates [for example, M. A. Hall and J. S. Lanchbury, Hum. Immunol. 43, 207 (1995); T. Nanki, H. Kohsaka, N. Miyasaka, J. Immunol. 161, 228 (1998)].
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Nanki, T.1
Kohsaka, H.2
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Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 2
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32p]-adenosine-5′-triphosphate-labeled oligonucleotides, as recommended by Amersham Pharmacia Biotech and by J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, ed. 2, 1989). The oligonucleotides were 20 to 22 mers, each with melting temperature of 64°C. The first oligonucleotide mix consisted of 12 oligonucleotides and, in control experiments, hybridized to 87% of a mix of bacterial clones known to bear a relevant insert. We also tested two relevant clones separately, and in both cases, the mix hybridized to >90% of colonies, although the strength of the signal was not uniform. Of 28 different clones with unrelated CDR3 sequences, none gave a positive signal. The second mix consisted of five oligonucleotides. In random sequencing, 19% of the clones in the first sample bore relevant inserts; the second oligonucleotide mix gave a detectable signal in 10% of the clones. We identified one relevant clone not detected by the mix, and none of 16 irrelevant clones were positive.
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(1989)
Molecular Cloning
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Sambrook, J.1
Fritsch, E.F.2
Maniatis, T.3
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15
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0344816313
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V. Baron and T. P. Arstila, data not shown
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V. Baron and T. P. Arstila, data not shown.
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16
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0344384610
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note
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+ cells, the purity was 95%.
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17
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0345679088
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note
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β profile.
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18
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P. Borgulya, H. Kishi, Y. Uematsu, H. von Boehmer, Cell 69, 529 (1992); C. J. MacMahan and P. J. Fink, Immunity 9, 637 (1998); E. Padovan et al., Science 262, 422 (1993).
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note
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We thank P. Bousso, A. Cumano, A. Vaheri, O. Vapalahti, and M. Viljanen for comments on the manuscript. Supported by the European Molecular Biology Organization and Academy of Finland (T.P.A.) and Ligue Nationale Contre le Cancer.
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