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 introdu ction of methods in which the length heterogeneity of the third comple mentarity region (CDR3) is used to further refine V-family-specific PC R. 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 metho dologies are cumbersome in the number of PCR reactions and gels needed for complete analysis of TCR BV repertoires. We describe here the opt imized conditions for using 11 TCR BV primer pairs in multiplex PCR wh ich allow for a more compact analysis. In addition, the two primers ac t as controls for each other in the PCR. The use of these primers is s hown using either fluorescent or radiolabeled constant primers. The tw o labeling methods give comparable results. Fluorescent primers avoid the difficulties associated with use of radioactivity, Autoradiography with P-32-labeled primers is simpler, requiring less instrumentation.