Kuhn David N, Borrone James, Meerow Alan W, Motamayor Juan C, Brown J Steven, Schnell Raymond J
Department of Biological Sciences, Florida International University, Miami, FL 33199, USA.
Electrophoresis. 2005 Jan;26(1):112-25. doi: 10.1002/elps.200406106.
We investigated the reliability of capillary array electrophoresis-single strand conformation polymorphism (CAE-SSCP) to determine if it can be used to identify novel alleles of candidate genes in a germplasm collection. Both strands of three different size fragments (160, 245 and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temperatures (18 degrees C, 25 degrees C, 30 degrees C, and 35 degrees C). Mixtures of amplified fragments of either the intron interrupting the C-terminal WRKY domain of the Tc10 locus or the NBS domain of the TcRGH1 locus of Theobroma cacao were electroinjected into all 16 capillaries of an ABI 3100 Genetic Analyzer and analyzed three times at each temperature. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments. A statistical analysis of the means of the fragment mobilities demonstrated that single-stranded conformers of the fragments could be reliably identified by their mobility at all temperatures and size classes. The order of elution of fragments was not consistent over strands or temperatures for the intermediate and large fragments. If samples are only run once at a single temperature, small fragments could be identified from a single strand at a single temperature. A combination of data from both strands of a single run was needed to identify correctly all four of the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large fragments that differed by only a single single-nucleotide polymorphism (SNP) from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be < or =250 bp, samples should be analyzed at two different temperatures between 18 degrees C and 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures, and undenatured double-stranded (ds)DNA molecular weight standards, such as ROX 2500, should be included as internal standards.
我们研究了毛细管阵列电泳-单链构象多态性（CAE-SSCP）的可靠性，以确定其是否可用于在种质库中鉴定候选基因的新等位基因。对三个不同大小片段（160、245和437 bp）的两条链进行了分析，这些片段在序列上相差一个或多个核苷酸，分析温度为四个不同温度（18℃、25℃、30℃和35℃）。将可可树Tc10基因座C端WRKY结构域内含子或TcRGH1基因座NBS结构域的扩增片段混合物电注入ABI 3100遗传分析仪的所有16根毛细管中，并在每个温度下分析三次。由于在这些实验中同时分析了中等大小和大的片段，因此不同大小范围的样品可以进行多重分析。对片段迁移率均值的统计分析表明，片段的单链构象体可以通过其在所有温度和大小类别下的迁移率可靠地鉴定出来。对于中等大小和大的片段，片段的洗脱顺序在链或温度上并不一致。如果样品仅在单一温度下运行一次，则可以在单一温度下从单链中鉴定出小片段。需要一次运行中两条链的数据组合才能正确鉴定所有四个中等片段，并且链或温度的数据组合都无法从一次运行中正确鉴定仅相差一个单核苷酸多态性（SNP）的两个大片段。因此，为了在毛细管阵列上使用SSCP充分评估候选基因座的等位基因，片段应≤250 bp，样品应在18℃至30℃之间的两个不同温度下进行分析，以减少毛细管引入的变异性，数据应来自两条链和两个温度的组合，并且应包括未变性的双链（ds）DNA分子量标准品，如ROX 2500，作为内标。
