Laboratory diagnosis of fragile X syndrome by using rapid cycle polymerase chain reaction

Fragile X syndrome in a common from of inherited mental retardation with an unusual inheritance pattern. The responsible mutation, a large increase in the number of CGG repeats flanking the fragile X mental retardation 1 (FMR-1) gene on the X chromosome, has recently been described. The polymerase chain reaction (PCR) has been used to amplify DNA across the region of CGG repeats to detect the mutation. However, standard procedures take 4-6 hours with low amplification efficiency for long mutated genes that prevent visualization with ethidium bromide staining on agorose gels. In this theses, factors that affect PCR amplification of the FMR-1 region have been systematically studied. These include 1) optimal concentration of MgCl2, dimethyl sulfoxide (DMSO), and 7-deaza-2'deoxyguanosine 5'-triphosphat (7-deaza-dGTP); 2) temperature control of the reaction mixture before the initiation of PCR; 3) denaturation temperature during PCR; and 4) polymerase of polymerase combination used. Optimal concentrations were 1.0 mM Mg2+ (wirh 200 upsilonM of each dNTP), 5-7.5% DNSo, and 0-25% 7-deaza-dGTP substitution of dGTP. A denaturation temperature of 97°C with a rapid temperature transition of the preamplification sample for 0° to 97°C enhanced amplification of high G+C products. Combination of Taq and Pfu polymerase, or KlenTaq1 and Pfu polymerases where Pfu polymerase is used as a minor component with 3' through 5' proofreading activity allow the complete extension of long amplification products from abnormal FMR-1 genes. With systematic optimization, genetic screening of at-risk populations for fragile X with PCR can be improved.