Detection of the hemoglobin E mutation using the color complementation
assay: application to complex genotyping
SH Embury, GL Kropp, TS Stanton, TC Warren, PA Cornett and FF Chehab
Department of Medicine, San Francisco General Hospital, University of
California, San Francisco 94110.
The color complementation assay (CCA) is a method of allele-specific DNA
amplification by which competitive priming and extension of fluorescently
labeled oligonucleotide primers determine the color of DNA amplification
product. This diagnostic method precludes the need for radioisotopes,
electrophoresis, and multiple high-stringency reaction conditions. The
multiplicity of mutant globin genes present in Southeast Asians complicates
clinical diagnosis and underscores the importance of DNA-based diagnostic
methods. We have applied CCA to distinguish beta A and beta E alleles.
Competing 15mer primers were a fluorescein-labeled complement to beta A and
a rhodamine-labeled complement to beta E, identical except for their
central nucleotides. A common unlabeled primer was used to amplify DNA
product, the color of which was determined by the perfectly complementary
primer. Color photography and spectrofluorometry, as well as a method of
black-white photography that we developed to distinguish fluorescein- and
rhodamine- labeled DNA, were used to record results. We applied CCA to
define the complex genotype of a Thai woman with thalassemia intermedia,
96% HbE, and 4% HbF whose possible genotypes included several permutations
of alpha-thalassemia, beta-thalassemia, and beta E genes. zeta-Globin gene
mapping of DNA doubly digested with Bg/II and Asp 718 showed the -alpha
3.7/--SEA genotype, and CCA confirmed homozygous beta E/beta E. The CCA is
useful for diagnosing the compound hemoglobin genotypes of Southeast Asians
and could be applied also to prenatal diagnosis in this population.
Volume 76,
Issue 3,
pp. 619-623,
08/01/1990
Copyright © 1990 by The American Society of Hematology
