Characterization of β-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA

TLDR: This work studied the genomic DNA of five patients with β-thalassaemia, and found two previously undescribed mutations, along with three known alleles, including the first natural mutation observed at the cap site of the β-globin gene.

Abstract: Direct sequencing of specific regions of genomic DNA1 became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA2–5. Recently, human mitochondria! DNA was amplified and directly sequenced6. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R. K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with β-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106–107 and the other is an A–C transversion at the cap site (+1) of the β-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.