The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA.
DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.
Diaz-Chico
J C
JC
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-3331.
Yang
K G
KG
Yang
K Y
KY
Efremov
D G
DG
Stoming
T A
TA
Huisman
T H
TH
D001483:Base Sequence; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D005091:Exons; D005784:Gene Amplification; D005796:Genes; D005914:Globins / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D009154:Mutation; D013789:Thalassemia / Q000097:blood / Q000235:genetics*
D005914:Globins; D004262:DNA Restriction Enzymes