Submitted to: Electrophoresis
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/19/2004
Publication Date: 1/19/2005
Citation: Kuhn, D.N., Borrone, J.W., Meerow, A.W., Motamayor, J., Brown, J.S., Schnell Ii, R.J. 2005. Single strand conformation polymorthism analysis of candidate genes for reliable identification of alleles by capillary array electrophoresis. Electrophoresis. 26:112-125. Interpretive Summary: Most DNA markers are separated based on length differences between alleles at a locus. However, many candidate genes are the same length but contain DNA sequence differences known as Single Nucleotide Polymorphisms (SNP). The detection of sequence differences in DNA fragments of the same length require a more technically demanding technique than the detection of differences based on length alone. One technique that allows this separation is Single Strand Conformation Polymorphism analysis (SSCP). Most other techniques for detecting SNP require extensive knowledge of the gene sequence among the experimental populations and are very expensive. We investigated the use of high throughput SSCP using a 16 capillary genetic analyzer to separate alleles in three size classes and at four different temperatures as an alternative to the more expensive SNP techniques. We determined that for alleles of 250 nucleotides or less we could reliably distinguish different alleles at the same locus using two different temperatures. For alleles larger than this, it is not possible to reliably identify different alleles. The utilization of automated high throughput SSCP for fragments less than 250 nt allows the discrimination of alleles at a locus without the extensive sequence knowledge required for SNP analysis.
Technical Abstract: We investigated the reliability of capillary array electrophoresis-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 bp, 245 pb and 437 bp) that differed by one or more nucleotides in sequence were analyzed at four different temepratures (18, 25, 30, and 35 degrees C). Mixtures of cloned alleles were electroinjected into all 16 capillaries of an ABI3100 Genetic Analzer and analyzed three times at each temperature. A statistical analysis of the means of the allele mobilities demonstrated that single stranded conformers of the alleles could be reliably identified by their mobility at all temperatures and size classes. The order of elution of alleles 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 alleles for the intermediate fragments and no combination of data from strands or temperatures would allow the correct identification of two large alleles that differed by only a single SNP from a single run. Thus, to adequately assess alleles at a candidate gene locus using SSCP on a capillary array, fragments should be greater or equal to 250 bps, samples should be analyzed at two different temperatures between 18 to 30 degrees C to reduce the variability introduced by the capillaries, data should be combined from both strands and both temperatures and undenatured dsDNA molecular weight standards, such as ROX2500, should be included as internal standards. Multiplexing of samples of different size range is possible, as intermediate and large fragments were analyzed simultaneously in these experiments.