Abstract
The detection of molecular defects of hemoglobin variants using mutagenically separated polymerase chain reaction (MS-PCR) was applied in this study. Using different lengths of allele-specific mutagenic primers, normal and mutant alleles of hemoglobin genes were amplified in the same reaction tube. Subsequent gel electrophoresis showed at least one of the two allelic products at the same loci or at least two of the several allelic products at different loci. We employed MS-PCR to test the following hemoglobin variants: Hb Constant Spring (Hb CS), Hb E, Hb G-Taichung, Hb J-Meinung, and Hb Kaohsiung. The results were the same as those obtained by amplified created reaction sites (ACRS) or direct sequencing. We conclude that the MS-PCR provides a rapid and simple alternative to other techniques for mutation detection in hemoglobin variants. Moreover, the principle can be extended to other genetic diseases.
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References
Barany F (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci U S A 88:189–193
Bunn HF, Forget BG (1986) The thalassemias: molecular pathogenesis and human hemoglobin variants. In: Hemoglobin: molecular, genetic and clinical aspects. W.B. Saunders, Philadelphia, pp 223–321, 381–451
Cai SP, Kan YW (1990) Identification of the multiple Β-thalassemia mutations by denaturing gradient gel electrophoresis. J Clin Invest 85:550–553
Chang JG, Chen PH, Chiou SS, Lee LS, Perng LI, Liu TC (1992) Rapid diagnosis of Β-thalassemia mutation in Chinese by naturally and amplified created restriction sites. Blood 80:2092–2096
Chang JG, Tseng WP, Yang LH, Lee LS, Chen PH, Liu TC (1993) Rapid diagnosis of Hb Constant Spring and Hb E by amplified created restriction sites. Blood 81:565–566
Dianzani I, Camaschella C, Saglio G, Forrest SM, Ramus S, Cotton RG (1991) Simultaneous screening for Β-thalassemia mutations by chemical cleavage of mismatch. Genomics 11:48–53
Embury SH, Kropp GL, Stanton TS, Warren TC, Cornett PA, Chehab FF (1990) Detection of the hemoglobin E mutation using the color complementation assay: application to complex genotyping. Blood 76:619–623
Fairbanks VF, Gilchrist GS, Brimhall B, Jereb JA, Goldston EC (1979) Hemoglobin E trait reexamined: a cause of microcytosis and erythrocytosis. Blood 53:109–115
Fucharoen S, Winichagoon P (1987) Hemoglobinopathies in southeast Asia. Hemoglobin 11:65–88
Huisman THJ (1986) The hemoglobinopathies: methods in hematology, vol 15. Churchill Livingstone, Edinburgh
Liu TC, Yen JS, Shen JS, Chen YH, Lee LS, Chen PH, Chang JG (1992) Rapid molecular diagnosis of hemoglobin variants by RT-PCR of reticulocyte mRNA and direct sequencing. Hemoglobin 16:379–388
Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF (1989) Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 17:2503–2516
Orita M, Suzuki Y, Sekiya T, Hayashi K (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5:874–879
Rust S, Funke H, Assmann G (1993) Mutagenically separated PCR (MS-PCR): a highly specific one step procedure for easy mutation detection. Nucleic Acids Res 21:3623–3629
Saiki RK, Bugawan TL, Horn GT, Mullis KB, Erlich HA (1986) Analysis of enzymatically amplified Β-globin and HLA-DQα DNA with allele-specific oligonucleotide probe. Nature 324:163–166
White MB, Carvalho M, Derse D, O'Brien SJ, Dean M (1992) Detecting single base substitutions as heteroduplex polymorphism. Genomics 12:301–306
Wood N, Standen G, Hows J, Bradley B, Bidwell J (1993) Diagnosis of sickle-cell disease with a universal heteroduplex generator. Lancet 342:1519–1520
Wu DY, Ugozzoli L, Pal BK, Wallace RB (1989) Allele-specific enzymatic amplification of Β-globin genomic DNA for diagnosis of sickle cell anemia. Proc Natl Acad Sci U S A 86:2757–2760
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This study was supported in part by a grant from the National Science Council, Taiwan, ROC (Grant No. NSC 84-2331-B-196-001-M02)
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Chang, J.G., Chang, C.P., Lu, C.M. et al. Rapid detection of hemoglobin variants by mutagenically separate polymerase chain reaction (MS-PCR). Ann Hematol 71, 97–100 (1995). https://doi.org/10.1007/BF01699253
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DOI: https://doi.org/10.1007/BF01699253