Figure 2.
Reversion of somatic mutations. (A) PCR strategy to revert mutated Ig genes into their germline counterparts. Asterics indicate somatic mutations. PCR 1 amplifies a germline VH gene corresponding to the VH in the mutated clone with gene specific primers. Primers used in PCR 2 revert somatic mutations in the mutated clone. Homology of the PCR 2 forward primer to the reverse primer used in PCR 1 is indicated. The PCR 2 reverse primer is JH-specific and contains the SalI restriction site. PCR products 1 and 2 are fused via the homologous region (indicated) in a subsequent overlap PCR using the same 5′AgeI VH specific forward primer as in PCR 1 and the 3′SalI JH specific reverse primer used in PCR 2 to generate the complete germline VDJ sequence. IgL chain somatic mutations are reverted following the same principle. Overlap PCR products are cloned into the respective expression vectors.
(B) Partial sequence of a representative mutated IgH gene (top) starting in FWR3 and including the CDR3 region and the conserved JH tryptophan (W) – glycine (G) motif (FWR4). The corresponding unmutated germline configuration after successful reversion of somatic mutations as indicated on the bottom. Dots represent nucleotide identity. Dashes are non-identical nucleotides. Nucleotide exchanges and aa exchanges are indicated in bold. Germline nucleotide sequences of full-length VH3-23, D3-3 and JH4 genes are indicated in italics.