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

Authors


Dr M. Losekoot. Department of Human Genetics (MGC). Sylvius Laboratorium. Wassenaarseweg 72, 2333 ALLeiden, The Netherlands.

Abstract

Summary The analysis of polymerase chain reaction (PCR)-amplified β-globin DNA with allele-specific oligonucleotide (ASO) probes reveals a very heterogeneous spectrum of β-thalassaemia in the Netherlands. However about 20% of the β-thalassaemia mutations cannot be identified with this approach. The combination of specific amplification of certain regions of the β-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 β-thalassaemia mutations occurs.

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