Reverse dot-blot detection of the African-American beta-thalassemia mutations.
DNA-based diagnosis of the beta thalassemias provides accuracy to newborn screening genetic counseling, and prenatal diagnosis. However, the use of polymerase chain reaction (PCR)-based methods is challenged by the great number of different-beta-thalassemia mutations that exist even within defined ethnic groups. In this regard, the reverse dot-blot method offers a means of screening for several mutations with a single hybridization reaction. We have applied the reverse dot-blot method to the detection of the beta-thalassemia mutations of African-Americans. We used two biotin-labeled primer pairs in a duplex reaction to amplify and label two beta-globin target DNA fragments that encompass all known African-American beta-thalassemia mutations. The PCR products were denatured and hybridized to polyT-tailed, membrane-fixed, allele-specific probe pairs for the hemoglobin (Hb) S, Hb C, and 14 beta-thalassemia mutations and their corresponding wild-type sequences. Seven common mutations plus Hb S and Hb C were included on one diagnostic strip, and seven less common beta-thalassemia mutations were included on another strip. Carefully controlled, high stringency hybridization allowed accurate distinction of these alleles. Reverse dot-blot diagnosis of the less common beta-thalassemia mutations precludes the need for alternative, more technically challenging methods. This method provides a rapid, accurate method for diagnosis of beta thalassemia among African-Americans and other ethnic groups in which beta thalassemia occurs.
Sutcharitchan
P
P
Department of Medicine, Chulalongkorn Hospital, Chulalongkorn University, Bangkok, Thailand.
Saiki
R
R
Huisman
T H
TH
Kutlar
A
A
McKie
V
V
Erlich
H
H
Embury
S H
SH
D001483:Base Sequence; D044383:Black People / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005914:Globins / Q000235:genetics*; D006444:Hemoglobin C / Q000235:genetics; D006451:Hemoglobin, Sickle / Q000235:genetics; D006453:Hemoglobinopathies / Q000175:diagnosis* / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D017086:beta-Thalassemia / Q000175:diagnosis* / Q000235:genetics; D001741:Black or African American
D017931:DNA Primers; D006451:Hemoglobin, Sickle; D005914:Globins; D006444:Hemoglobin C