Universal PCR amplification of mouse immunoglobulin gene variable regions:the design of degenerate primers and an assessment of the effect of DNA polymerase 3 ' to 5 ' exonuclease activity

Degenerate primers were designed for PCR amplification of unknown mouse imm unoglobulin (Ig) light (L) and heavy (H) chain variable (V) genes. Each sub group of mouse Ig gene sequences [Kabat, E.A., Wu, T.T., Perry, H.H., Gotte sman, K.S., Foeller, C., 1991. Sequences of Proteins of Immunological Inter est, 5th edn. US Department of Health and Human Services, Public Health Ser vice, NIH.] was analyzed, and highly degenerate primers in the framework on e (FR1) region were designed. A single highly degenerate FR1 primer suffice d for the amplification of light chains; for heavy chains, a series of FR1 primers was used. At the same time, we assessed the effect of 3' to 5' exon uclease activity of DNA polymerase on the utilization of these degenerate p rimers. Using Tag polymerase, which lacks 3' to 5' exonuclease activity, we successfully amplified the Ig VL and VH genes expressed in more than a hun dred monoclonal hybridoma cell lines reactive against a phosphonamidate hap ten. Sequence analysis of the cloned VL and VH genes, 52 of each, showed th at they are derived from multiple germline families (10 of the 17 VL famili es and 9 of the 14 VH families) as recently defined [Martinez, C., Lefranc, M., 1998. The mouse (Mus musculus) immunoglobulin kappa variable (IGKV) ge nes and joining (IGKJ) segments. Exp. Clin. Immunogenet. 15, 184.]. The uni versality of our primers was also demonstrated by successful amplification of other mouse hybridoma cell lines that are specific to different antigens . (C) 2000 Elsevier Science B.V. All rights reserved.