Newer Developments in the Identification of β-Thalasseuia

Abstract

One of the easiest and most sensitive methods of detecting mutations in the β-globin gene leading to β-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5–10 μg of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3–10 days. We wish to report a method for detecting these mutations which involves 1 μg of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the β-globin gene. In each primer set, one primer is complementary to the β-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the β-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 β-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.