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J. Biol. Chem., Vol. 264, Issue 14, 7780-7783, May, 1989

A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions

S Fucharoen, G Fucharoen, P Fucharoen and Y Fukumaki
Research Laboratory for Genetic Information, Kyushu University, Fukuoka, Japan.

The beta-globin genes from a Thai patient compound heterozygous for beta-thalassemia and HbE disease were investigated. The 3.0-kilobase fragment containing the entire beta-globin gene was amplified by polymerase chain reaction, using Taq DNA polymerase followed by direct cloning of the amplified product into plasmid DNA. Sequence analysis of the thalassemia gene revealed only one base change, a C-A transversion within codon for an amino acid 35. This new mutation creates a premature terminator, TAA, an ochre codon, and results in a beta 0- thalassemia phenotype. The same result was obtained when this mutation was analyzed using a conventional cloning technique, direct sequencing of the amplified product, and hybridization with allele-specific oligonucleotide probes. No misincorporation was detected in the sequence analysis of the 3.0-kilobase insert of five clones of the amplified products obtained from genomic DNA of a normal individual. This approach is a rapid and accurate method for molecular cloning of the beta-globin gene and also other genes, the partial nucleotide sequences of which are known.
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