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Asymmetrically primed selective amplification/temperature shift fluorescence polymerase chain reaction to detect the
G L Kropp1, S Fucharoen, S H Embury
1Department of Medicine, University of California San Francisco, San Francisco General Hospital 94110.
Blood
|July 1, 1991
Summary
A new DNA test accurately diagnoses Hemoglobin Constant Spring, a common cause of severe alpha-thalassemia in Southeast Asians. This polymerase chain reaction method offers a faster, more reliable diagnostic tool for this genetic blood disorder.
Area of Science:
- Molecular Biology
- Genetics
- Hematology
Background:
- Hemoglobin (Hb) Constant Spring is a prevalent alpha-thalassemic hemoglobinopathy in Southeast Asia, often leading to severe alpha-thalassemia.
- Conventional electrophoretic methods present diagnostic challenges for Hb Constant Spring due to high homology between alpha 2- and alpha 1-globin genes.
Purpose of the Study:
- To develop a novel DNA-based diagnostic method for Hemoglobin Constant Spring.
- To overcome limitations of existing diagnostic techniques, particularly the challenge of distinguishing between highly homologous globin genes.
Main Methods:
- Development of a single-reaction polymerase chain reaction (PCR) assay utilizing asymmetric priming and a temperature shift.
- The method selectively amplifies the alpha 2-globin gene, the site of the Hb Constant Spring mutation, while suppressing alpha 1-globin gene amplification.
- Allele-specific fluorescence PCR is employed for discriminating between normal and Hb Constant Spring alpha 2 genes.
Main Results:
- The developed PCR method successfully achieved selective amplification of the alpha 2-globin gene.
- Allele-specific fluorescence PCR accurately discriminated between normal and Hb Constant Spring alpha 2 gene sequences.
- The assay avoids radioisotopes and eliminates the need for electrophoresis, serving as its own amplification control.
Conclusions:
- This novel PCR-based method provides a sensitive and specific diagnostic tool for Hemoglobin Constant Spring.
- The technique offers significant advantages over previous methods, including simplicity, speed, and suitability for routine clinical use.
- The assay is readily applicable to routine diagnosis, population screening, and prenatal diagnosis of Hb Constant Spring.