Summary
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.
Similar content being viewed by others
Article PDF
References
Beutler E, Kuhl W (1990): The NT 1311 polymorphism of G6PD: G6PD Mediterranean mutation may have originated independently in Europe and Asia. Am J Hum Genet47: 1008–1012
Breslauer KJ, Frank R, Blocker H, Marky KA (1986): Predicting DNA duplex stability from the base sequence. Proc Natl Acad Sci USA83: 3746–3750
Ganczakowski M, Town M, Bowden DK, Vulliamy TJ, Kaneko A, Clegg JB, Weatherall DJ, Luzzatto L (1995): Multiple glucose 6-phosphate dehydrogenase-deficient variants correlate with malaria endemicity in the Vanuatu Archipelago (Southwestern pacific). Am J Hum Genet56: 294–301
Hirono A, Miwa S, Fujii H, Ishida F, Yamada K, Kubota K (1994): Molecular study of eight Japanese cases of glucose-6-phosphate dehydrogenase deficiency by nonradioisotopic single-strand conformation polymorphism analysis. Blood83: 3363–3368
Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989): Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci USA86: 2766–2770
Saha S, Saha N, Tay JSH, Jeyaseelan K, Basair JB, Chew SE (1994): Molecular characterisation of red cell glucose-6-phosphate dehydrogenase deficiency in Singapore Chinese. Am J Hematol42: 273–277
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA (1986): Analysis of enzymatically amplifiedβ-globin and HLA-DQa DNA with allele-specific oligonucleotide probes. Nature324: 163–165
Scholz RB, Milde-Langosch K, Jung R, Schlechte H, Kabisch H, Wagener C, Loning T (1993): Rapid screening for Tp53 mutations by temperature gradient gel electrophoresis: a comparison with SSCP analysis. Hum Mol Genet2: 2155–2158
Sheffield V, Cox D, Lerman L, Myers R (1989): Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc Natl Acad Sci USA86: 232–236
Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989): Allele-specific enzymatic amplification ofβ-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci USA86: 2757–2760
Yoshino K, Nishigaki K, Husimi Y (1991): Temperature sweep gel electrophoresis: a simple method to detect point mutations. Nucleic Acids Res19: 3153
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Shirakawa, T., Nishiyama, K., Poh-San, L. et al. A comprehensive method to scan for point mutations of the glucose 6 phosphate dehydrogenase gene. Jap J Human Genet 42, 417–423 (1997). https://doi.org/10.1007/BF02766942
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02766942
Key Words
This article is cited by
-
Molecular Heterogeneity of Glucose-6-Phosphate Dehydrogenase Deficiency in Malays in Malaysia
International Journal of Hematology (2002)