Delmotte F, Leterme N, Simon J C
UMR Biologie des Organismes et des Populations Appliquée à la Protection des Plantes, INRA, Le Rheu, France.
Biotechniques. 2001 Oct;31(4):810, 814-6, 818.
By comparing data collected with different automated sequencers and a manual technique (fragment separation in a silver-stained polyacrylamide gel), we found strong discrepancies in allele size of microsatellite loci. To quantify the sizing bias generated by automated capillary electrophoresis, we typed 51 alleles at seven loci andfound that differences between actual (manual) and called (automated) sizing were inversely related to locus size. This result seems independent of the fluorescent dye but might be due to different migration patterns of the size standard and the microsatellite loci. Thus, it is essential to distinguish between actual (that can only be confirmed by sequencing) and called (obtained with automated sequencer) allele sizes. To enable the comparison of data collected by different laboratories on different instruments, the greatest attention should be paid to material and protocol descriptions used for allele sizing, and reference standard DNA genotypes should be shared between collaborating laboratories. Without these precautions, scoring errors in allele size might result in important misleading conclusions.
通过比较使用不同自动测序仪和手动技术（银染聚丙烯酰胺凝胶中的片段分离）收集的数据，我们发现微卫星基因座的等位基因大小存在很大差异。为了量化自动毛细管电泳产生的大小偏差，我们对7个基因座的51个等位基因进行了分型，发现实际（手动）大小与测定（自动）大小之间的差异与基因座大小呈负相关。这一结果似乎与荧光染料无关，但可能是由于大小标准品和微卫星基因座的迁移模式不同所致。因此，区分实际（只能通过测序确认）和测定（通过自动测序仪获得）的等位基因大小至关重要。为了能够比较不同实验室在不同仪器上收集的数据，应特别注意用于等位基因大小测定的材料和方案描述，合作实验室之间应共享参考标准DNA基因型。如果没有这些预防措施，等位基因大小的评分错误可能会导致重要的误导性结论。
