Rapid diagnosis of β-thalassemia mutations in Chinese by naturally and amplified created restriction sites

Abstract

We developed a rapid and simple method to diagnose the molecular defects of β-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human β globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41-42 we introduced a single mismatch nucleotide into the 3′ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C → T) of IVS-2, an Alu I restriction site was created to detect the codon 17 mutation (A → T), and EcoRI restriction site was created for the -28 mutation (A → G), a Rsa I restriction site was created for the nucleotide 5 mutation (G → C) of IVS-1 and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of β-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis. © 1992 by The American Society of Hematology.