Rapid differentiation of five common alpha-thalassemia genotypes by polymerase chain reaction.
The alpha-thalassemias are common genetic disorders that arise from reduced synthesis of the alpha-globin chains. At present, large-scale carrier screening and clinically valuable antenatal detection programs have not been established for the congenital disorder alpha-thalassemia (alpha-thal). We have developed a simple nonradioactive polymerase chain reaction (PCR) approach that can detect and differentiate several common alpha-globin gene deletional alpha-thals regardless of the break points. When three primer sets were used--two gene-specific sets for the alpha1- and alpha2-globin genes and one set for the beta-actin gene (serving as an internal control)--PCR products from genomic DNA were simultaneously amplified and analyzed after coamplification and gel electrophoresis. The number of alpha-globin genes present in the subjects was determined by the intensity of alpha1 and alpha2 bands normalized with that of beta-actin when using densitometry. Our results demonstrate that five common genotypes of deletional alpha-thal are differentiated by the ratios of alpha1/beta-actin and alpha2/beta-actin. We also examined the feasibility of coupling this allele-specific amplification to a color-complementary assay. This easy and reproducible PCR assay is suitable for identifying alpha-thal carriers in screenings of large populations and improving genetic counseling.
Molecular and Clinical Hematology Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA
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DC
Tang
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Fucharoen
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Ding
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Rodgers
D000199:Actins / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers; D005838:Genotype; D005914:Globins / Q000235:genetics; D006801:Humans; D016133:Polymerase Chain Reaction; D017085:alpha-Thalassemia / Q000235:genetics*
D000199:Actins; D017931:DNA Primers; D005914:Globins