IMPROVED SIZING OF FRAGILE-X CCG REPEATS BY NESTED POLYMERASE CHAIN-REACTION

We have developed an improved method for polymerase chain reaction (PC R)-based sizing of the CCG repeat region at the fragile X locus, FMR-1 . This method is designed to optimize denaturation and replication of long repeats with high G + C content, which are otherwise refractory t o amplification. The method utilizes nested PCR primers to increase se nsitivity and specificity. Alkaline denaturation of the genomic templa te DNA, combined with addition of glycerol and deaza-dGTP, facilitates strand separation. Labeled PCR products are sized on denaturing polya crylamide gels. For alleles in the normal-to-premutation size range, s trong reproducible signals are routinely obtained from small amounts o f rapidly prepared DNA. This allows precise determination of the CCG r epeat number, providing data related to the expansion potential of the repetitive segment. Detection of large premutations and some full mut ations is also enhanced by the improved procedure. (C) 1994 Wiley-Liss , Inc.