Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis.
A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino)ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25 degrees C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2 degrees C/min and were induced externally. A total of 24 samples from affected patients, both in the homo- and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples.
Gelfi
Cecilia
C
CNR, INB, L.I.T.A., Segrate, Milano, Italy. gelfi@itba.mi.cnr.it
Vigano
Agnese
A
De Palma
Sara
S
Righetti
Pier Giorgio
PG
Righetti
Sabin Carla
SC
Corna
Elisabetta
E
Zunino
Franco
F
D017738:Alkanesulfonic Acids; D002021:Buffers; D002482:Cellulose / Q000031:analogs & derivatives*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009025:Morpholines; D009930:Organic Chemicals; D017354:Point Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D017738:Alkanesulfonic Acids; D002021:Buffers; D005456:Fluorescent Dyes; D009025:Morpholines; D009930:Organic Chemicals; C098798:SYBR Green II; D016159:Tumor Suppressor Protein p53; C004550:2-(N-morpholino)ethanesulfonic acid; D002482:Cellulose; C002283:hydroxyethylcellulose