Antibody repertoire development in fetal and neonatal piglets. V. VDJ gene chimeras resembling gene conversion products are generated at high frequency by PCR in vitro.
The recovery of VDJ rearrangement is most often accomplished by PCR amplification of DNA extracted from mixtures of B-cells. Using this procedure in swine, VDJs containing chimeric V(H) genes that resemble gene-conversion products, are frequently encountered. To examine whether these chimeras could be the result of PCR artifacts, we used different combinations of swine VDJ templates, each having unique CDR1, CDR2 and D(H) segments, to generate >2600 clones. Using equal amounts of two templates and 30 cycles of PCR, up to 45% of the resultant clones were VDJ chimeras. The frequency of chimeras was independent of the specific VDJ template and the chimeras were generated regardless of whether Taq-, Pfu- or mixtures of Taq- and Pfu-polymerases were employed or whether PCR extension time was prolonged six-fold. The frequency of generating chimeras was dependent on the ratio of the two target DNAs although even ratios approximately 1:10 generated approximately 10% chimeric VDJs. Chimeras could be generated using only 10 cycles of PCR or using the initial template DNAs diluted as much as 1:10000. Of the 279 chimeric VDJs generated, 61% of the crossovers occurred in FR3, 21% in FR2 and 18% in both FR2 and FR3. We interpret these results to mean that in vivo gene conversion in this species can only be unambiguously proven when the VDJs from individual B-cells are bearing a single VDJ rearrangement amplified and sequenced or when VDJs are cloned without the use of PCR.
Sinkora
M
M
Department of Microbiology and Interdisciplinary Immunology Program, 3-501L Bowen Science Building, 51 Newton Road, The University of Iowa, 52242, Iowa City, IA, USA. marek-sinkora@uiowa.edu
Sun
J
J
Butler
J E
JE
D000818:Animals; D000831:Animals, Newborn / Q000276:immunology; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D005314:Embryonic and Fetal Development / Q000276:immunology; D015322:Gene Rearrangement, B-Lymphocyte / Q000276:immunology*; D005803:Genes, Immunoglobulin; D016133:Polymerase Chain Reaction; D013552:Swine
D000906:Antibodies