PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts.
Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.
Zylstra
P
P
Department of Biological Sciences, University of Wollongong, New South Wales, Australia.
Rothenfluh
H S
HS
Weiller
G F
GF
Blanden
R V
RV
Steele
E J
EJ
D000818:Animals; D004587:Electrophoresis, Agar Gel; D005260:Female; D005796:Genes / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D005810:Multigene Family / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic / Q000235:genetics
D007135:Immunoglobulin Variable Region