Accurate determination of the number of CAG repeats in the Huntington disease gene using a sequence-specific internal DNA standard

We have developed a sequence-specific internal DNA size standard for the ac curate determination of the number of CAG repeats in the Huntington disease (HD) gene by cloning key fragments (between 15 and 64 CAG repeats) of the HD gene. These fragments, pooled to produce a sequence-specific DNA ladder, enabled us to observe the true number of CAG repeats directly, with no nee d for calculations. Comparison of the calculated numbers of CAG repeats in the HD gene using this sequence-specific DNA standard with a commercially a vailable standard (GENESCAN-500 TAMRA) showed that the latter underestimate d the number of CAG repeats by three when analyzed by capillary electrophor esis on the ABI 310 Genetic Analyzer (POP4 polymer). In contrast, the use o f the same standard overestimated the number of CAG repeats by one when the samples were analyzed by denaturing polyacrylamide electrophoresis on ABI 377 DNA Sequencer (6% denaturing polyacrylamide gel). This suggests that ou r sequence-specific standard provides greater accuracy for the determinatio n of the true number of CAG repeats in the HD gene than commercially availa ble standards. The sequence-specific standard can be radioactively labeled and successfully replace conventional DNA size standards when analyzing pol ymerase chain reaction (PCR)-amplified HD alleles by denaturing polyacrylam ide electrophoresis.