Molecular analysis of T cell repertoires. Spectratypes generated by multiplex polymerase chain reaction and evaluated by radioactivity or fluorescence.
The analysis of T-cell repertoires has been facilitated by the introduction of methods in which the length heterogeneity of the third complementarity region (CDR3) is used to further refine V-family-specific PCR. We call our implementation of this technique T-cell spectratyping. This method is especially important in analysis of specific expansion or retention of T cells in human immune system function. Current methodologies are cumbersome in the number of PCR reactions and gels needed for complete analysis of TCR BV repertoires. We describe here the optimized conditions for using 11 TCR BV primer pairs in multiplex PCR which allow for a more compact analysis. In addition, the two primers act as controls for each other in the PCR. The use of these primers is shown using either fluorescent or radiolabeled constant primers. The two labeling methods give comparable results. Fluorescent primers avoid the difficulties associated with use of radioactivity. Autoradiography with 32P-labeled primers is simpler, requiring less instrumentation.
Maślanka
K
K
Immunogenetic Research Section, Blood Center of Southeastern Wisconsin, Milwaukee 53233, USA.
Piatek
T
T
Gorski
J
J
Yassai
M
M
Gorski
J
J
D001345:Autoradiography; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D013964:Thyroid Neoplasms / Q000097:blood / Q000276:immunology / Q000473:pathology
D017931:DNA Primers; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D011948:Receptors, Antigen, T-Cell