Site-directed mutagenesis of exon 5 of p53: purification, analysis, and validation of amplicons for DHPLC.
Reversed-phase ion-pair chromatography is used in its three modes of operation--(1) non-denaturing, (2) partially denaturing, (3) fully denaturing - to isolate and characterize five site-directed mutant amplicons of exon 5 of the p53 gene (GenBank X54156). The mutant fragments were prepared using a simplified mutagenesis procedure without cloning and isolated in high purity under non-denaturing conditions. Then, under partially denaturing conditions, heteroduplices formed by hybridization of each of the five mutants with wild-type were all detected by denaturing reversed-phase high-performance chromatography (DHPLC). The fidelity of the PCR step was also assessed. Finally, the homogeneity of each mutant was confirmed by minisequencing under fully denaturing conditions. The versatility and facility of the method for DNA manipulations was exploited to demonstrate a strategy for validating amplicons for mutation detection by DHPLC.
Narayanaswami
Gowri
G
Transgenomic Inc., San Jose, CA 95131, USA.
Taylor
Paul D
PD
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification*; D005091:Exons; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D016159:Tumor Suppressor Protein p53; D004247:DNA