Figure 3.

Amplification of V_HH fragments by high-coverage hinge-specific primers.A, schematic representation of hinge primers selected for higher coverage single-step PCR amplification of V_HH regions. Forward primer CALL001 anneals in the leader region upstream of FR1, while reverse primers LH-rev and SH-rev anneal in the long-hinge or short-hinge region, respectively, downstream of FR4. In contrast, second step nested primers VHH-Fwd and VHH-Rev anneal in FR1 or FR4 regions. B, high-throughput sequencing of lymphocyte mRNA was used to determine what percentage of V_HH-encoding transcripts could be recognized by primers used for traditional nested PCR (blue) or hinge-targeted PCR (yellow) by searching each primer against the total sequence database. C, Venn diagram comparing the number of unique FR4 sequences captured using each primer set, as determined by adjacent sequence in V_HH transcript. New hinge primers correspond to a wider diversity of FR4 sequences. Incomplete sequencing coverage, FR4 mismatches from “old” V_HH primers, and amplification of non-IgG sequences contribute to nonoverlapping sequences.