Capillary electrophoretic analysis of DNA restriction fragments and PCR products for polymorphism and mutation studies in cystic fibrosis and Gaucher's disease.
Two methods are described for the analysis of DNA restriction fragments and PCR products in studies on polymorphism and mutation in cystic fibrosis and Guacher'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 degrees 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 microns i.d., with Tris-borate buffer containing urea. Typical electropherograms are presented for the analysis of CF mutation delta F508 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 x 100 microns i.d.(ABSTRACT TRUNCATED AT 250 WORDS)
Bory
C
C
Centre d'Etudes des Maladies Métaboliques, Hôpital Debrousse, Lyon, France.
Chantin
C
C
Bozon
D
D
D003550:Cystic Fibrosis / Q000235:genetics*; D004247:DNA / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons / Q000502:physiology; D005776:Gaucher Disease / Q000235:genetics*; D006801:Humans; D007202:Indicators and Reagents; D007962:Leukocytes / Q000737:chemistry; D009154:Mutation; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping
D007202:Indicators and Reagents; D004247:DNA