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

被引:69
作者
Zylstra, P
Rothenfluh, HS
Weiller, GF
Blanden, RV
Steele, EJ
机构
[1] Univ Wollongong, Dept Biol Sci, Mol Immunol Lab, Wollongong, NSW 2522, Australia
[2] Australian Natl Univ, John Curtin Sch Med Res, Div Immunol & Cell Biol, Canberra, ACT 2601, Australia
[3] Australian Natl Univ, Res Sch Biol Sci, Bioinformat Lab, Canberra, ACT 2601, Australia
关键词
artefact; immunoglobulin variable gene; PCR; recombination;
D O I
10.1046/j.1440-1711.1998.00772.x
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
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 Tag 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 similar to 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.
引用
收藏
页码:395 / 405
页数:11
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