title: Asymmetrically primed selective amplification/temperature shift fluorescence polymerase chain reaction to detect the hemoglobin Constant Spring mutation.
abstract: Hemoglobin (Hb) Constant Spring is an alpha-thalassemic hemoglobinopathy that is a major cause of severe alpha-thalassemia in Southeast Asians. The difficulty of diagnosing Hb Constant Spring using standard electrophoretic methods has led to interest in DNA-dependent diagnostic methods. The methods developed have had to contend with the high degree of homology of the alpha 2-globin gene (the site of the Hb Constant Spring mutation) and the alpha 1-globin gene. We have developed a single reaction polymerase chain reaction-based method that uses asymmetric priming and a temperature shift to accomplish dual ends, selective amplification of alpha 2 but not alpha 1 DNA and discrimination of normal and Hb Constant Spring alpha 2 genes by allele-specific fluorescence polymerase chain reaction. Advantages of this method over previous approaches include avoiding radioisotopes, precluding the need for electrophoresis, and serving as its own control for successful amplification. It is readily applicable to routine diagnosis, population screening, and prenatal diagnosis.
authors.affiliation: Department of Medicine, University of California San Francisco, San Francisco General Hospital 94110.
authors.forename: G L
authors.initials: GL
authors.lastname: Kropp
authors.forename: S
authors.initials: S
authors.lastname: Fucharoen
authors.forename: S H
authors.initials: SH
authors.lastname: Embury
mesh_terms: D000483:Alleles; D000510:Alpha-Globulins / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005453:Fluorescence; D005784:Gene Amplification / Q000235:genetics; D006455:Hemoglobins, Abnormal / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013789:Thalassemia / Q000235:genetics
chemical_list: D000510:Alpha-Globulins; D006455:Hemoglobins, Abnormal; D004247:DNA; C035541:Hemoglobin Constant Spring