PCR amplification of murine immunoglobulin germline V genes: Strategies for minimization of recombination artefacts

Paula Zylstra, Harald S. Rothenfluh, Georg F. Weiller, Robert V. Blanden, Edward J. Steele*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    76 Citations (Scopus)

    Abstract

    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 ~ 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.

    Original languageEnglish
    Pages (from-to)395-405
    Number of pages11
    JournalImmunology and Cell Biology
    Volume76
    Issue number5
    DOIs
    Publication statusPublished - 1998

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