Problem With Microsatellite Polymerphism Analysis and its Practical Solution Detection of Complex STR Alleles by Single-Strand Conformation Polymorphism (SSCP) Analysis and Denaturing Gradient Gel Electrophoresis (DGGE)

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We amplified the complex short tandem repeat (STR) loci D11S488 and D11S554, electrophoresed amplicons on denaturing polyacrylamide gels, and detected them by Southern blotting. Sequencing of 262 bp-long D11S488 alleles revealed five different alleles with structural variations. Sequence analysis of the D115554 locus showed that 237 bp, 225 bp and 217 bp alleles had three, two, and two different sequences, respectively.Single-strand conformation polymorphism (SSCP) analysis of D115488 amplicons on a 6% native polyacrylamide gel at 4ﾟC enabled us to distinguish four alleles of the same length. Similarly, we were able to identify different D11S554 alleles of the same length by electrophoresis on a 5% polyacrylamide gel at 15ﾟC.We successfully distinguished four different D115488 alleles (262 bp) by 20-50% denaturing gradient gel electrophoresis (DGGE), and all of the different D115554 alleles mentioned above by 25-35% DGGE.However, some alleles of different lengths showed the same mobility.We applied the SSCP technique to two normal trio cases of disputed parentage, in which four STR loci including D115488 were tested for. Both trios had an apparently identical 262 bp D115488 allele on a denaturing gel. In one case, SSCP analysis increased the combined probability of paternity, ' because the child and the alleged father shared a rare allele. In the other case, the child shared the allele with the mother, but not with the alleged father, which excluded paternity. As a result, paternity was excluded by three of the four loci tested.Allowing simple and rapid detection of sequence polymorphisms, our method is suited for practical use.