Denaturing gradient gel electrophoresis and direct sequencing of PCR amplified genomic DNA: a rapid and reliable diagnostic approach to beta thalassaemia

Br J Haematol. 1990 Oct;76(2):269-74. doi: 10.1111/j.1365-2141.1990.tb07883.x.

Abstract

The analysis of polymerase chain reaction (PCR)-amplified beta-globin DNA with allele-specific oligonucleotide (ASO) probes reveals a very heterogeneous spectrum of beta-thalassaemia in the Netherlands. However about 20% of the beta-thalassemia mutations cannot be identified with this approach. The combination of specific amplification of certain regions of the beta-globin gene with denaturing gradient gel electrophoresis (DGGE) allowed us to rapidly localize several of these mutations to specific regions of the gene, which were again amplified and directly sequenced. We believe that the combination of DGGE and the direct sequence determination of PCR amplified genomic DNA represents a valid alternative to the 'ASO probes' approach, especially in countries where a very heterogeneous spectrum of beta-thalassaemia mutations occurs.

MeSH terms

  • Base Sequence
  • Codon / genetics
  • DNA / genetics*
  • DNA / isolation & purification
  • Electrophoresis, Polyacrylamide Gel / methods
  • Genetic Carrier Screening*
  • Globins / genetics*
  • Humans
  • Molecular Sequence Data
  • Oligonucleotide Probes
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Genetic
  • Reference Values
  • Thalassemia / diagnosis*
  • Thalassemia / genetics

Substances

  • Codon
  • Oligonucleotide Probes
  • Globins
  • DNA