Capillary electrophoretic analysis of DNA restriction fragments and PCR products for polymorphism and mutation studies in cystic fibrosis and Gaucher's disease

Abstract

Two methods are described for the analysis of DNA restriction fragments and PCR products in studies on polymorphism and mutation in cystic fibrosis and Gaucher's disease, based on capillary electrophoresis. In one CE system, a Beckman kit for producing a chemical gel (polymerized within the capillary) is used for single-stranded DNA fragments from 10 to 300 bases in size. Its performance was demonstrated on the separation of a mixture of polydeoxyadenylic acids p(dA) 40–60 at 30°C. Electrokinetic injection was used (5–7 kV for 5–20 s), the applied field being 300 V cm −1 for an effective length of 7, 20 or 30 cm and 100 μm i.d., with Tris-borate buffer containing urea. Typical electropherograms are presented for the analysis of CF mutation ΔF508 in PCR products from homozygous and heterozygous individuals, illustrating the resolution of two complementary single strands (95b and 95b) of a DNA fragment. DNA fragments differing in size by only one base could also be resolved, as shown for the 105b and 106b fragments obtained from a heterozygote for 3905 insT CF mutation, with a run time of ca 45 min. If discrimination were only required between fragments differing by two or more bases, run times could be reduced by 6 when using a capillary length of only 7 cm × 100 μm i.d. A second CE system based on a kit for producing a physical gel (dissolution of polyacrylamide in buffer prior to filling the capillary) gave high resolution for double-stranded DNA fragments from 100 to 1500 base pairs under similar CE conditions, but with 175 V cm −1 at 20°C. This was shown for the DNA standard Phi-X 174 when DNA fragments differing in size by 5 base pairs could be resolved within the region 100–200 bp.