Efficiency and cost effectiveness: PAGE-SSCP versus MDE and Phast gels for the identification of unknown beta thalassaemia mutations

Abstract

Background: Prenatal diagnosis for beta thalassaemia has proved to be very effective in preventing the birth of an affected child and hence in controlling the disease. The success of prenatal diagnosis depends on the delineation of the underlying mutations in the population at risk. Each population carries a limited number of frequent defects (89-91%) and a variable number of rare alleles (4-5%), whereas 2-3% of alleles remain uncharacterised. To offer prenatal diagnosis when the parental mutation is unknown, the application of a non-specific detection method (such as single stranded conformational polymorphism (SSCP)) to localise the mutation, followed by direct sequencing of the amplified gene sequence, is required. With this objective in mind, this study was designed to devise the best protocol and system of SSCP for the rapid screening of unknown mutations in the beta globin gene.

Methods: To detect mutations in this disease, three different systems-Phast gels, MDE gels, and polyacrylamide gels-were used under varying conditions.

Results: Polyacrylamide gels were found to be the most efficient, both in terms of resolution and cost.

Conclusion: Polyacrylamide gels are the most rapid, efficient, reliable, and cost effective means for DNA mutation analysis of the beta globin gene.

Figure 1

A schematic representation of the human β globin gene with the location of the primers, and the region and the size (bp) of the fragments they amplify. IVS, intervening sequence (intron).

Figure 2

Autoradiograph of the Phast gel. Lanes 1 and 6, samples heterozygous for the CD 41/42 β thalassaemia mutation; lanes 2 and 3, and 5, samples homozygous for the CD 41/42 β thalassaemia mutation; lane 4, normal.

Figure 3

Autoradiograph of the MDE gel. Lanes 1 and 2, normal; lanes 3 and 4, samples heterozygous for the CD 41/42 β thalassaemia mutation.

Figure 4

Autoradiogram of the polyacrylamide single stranded conformational polymorphism gel run at constant 8 W for 16 hours at room temperature. Lanes 1 and 4, samples heterozygous for the CD 41/42 β thalassemia mutation; lanes 2 and 3, samples heterozygous for the CD 47/48 mutation.