TcR structure and cloning strategy.

<p>(a) TcRα and β mRNA structures are similar: The 5′-situated Variable (V) domain encodes for the N-terminal part of the extracellular domain (EC) of the TcR. It starts with the signal sequence (L) upstream of the V-region. It ends at the recombination site with the D domain (only in TcRβ) and Joining domain (J), representing the hypervariable CDR3 domain. On the 3′-side of the messenger lies the Constant (C) region, which encodes for the carboxy-terminal part of the EC, the transmembrane region (TM) and the short intracellular domain (IC). (b) The 5′-RACE of the TcR is performed by reverse-transcription of mRNA into cDNA using an oligo dT primer. The cDNA is then polyC-tailed by TdT and this reaction is followed by two sequential amplifications using nested primers (pC1 and pC2) together with a polyC annealing primer (pGI). The final product is cloned into pTA and sequenced. The sequenced portion results in the full length V-domain and the CDR3 if the tailing has occurred on the 5′-UTR.