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We have developed a fast and comprehensive method to scan for point mutations in a gene on X chromosome. A target region of the gene is first amplified. Then, using the amplified product as a template, PCR is carried out with multiple short-length forward primers arrayed in tandem in the scanned region, and a common reverse primer. The absence of amplified product defines the site of a mutation within a narrow region of the primer recognition site. To evaluate our method, point mutations in exon 12 of the human glucose-6-phosphate dehydrogenase (G6PD) gene were used as a model system. Out of 12 Singaporean G6PD-deficient patients, 6 cases were shown by the method to have a nucleotide change in this exon. Sequence analysis confirmed the presence of a nucleotide change in the region identified by our scanning. Thus, our method is accurate in localizing mutations within a narrow region, and allows large numbers of samples to be handled simultaneously.

A comprehensive method to scan for point mutations of the glucose 6 phosphate dehydrogenase gene