Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer.
Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP-CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2-(N-morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems.
Gelfi
C
C
CNR, Institute of Advanced Biomedical Technologies, Segrate (Milano), Italy. gelfi@itba.mi.cnr.it
Viganó
A
A
Curcio
M
M
Righetti
P G
PG
Righetti
S C
SC
Corna
E
E
Zunino
F
F
D002021:Buffers; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D019075:Electrophoresis, Capillary / Q000379:methods; D016158:Genes, p53; D006580:Genetic Carrier Screening / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
D002021:Buffers; D017931:DNA Primers; D004277:DNA, Single-Stranded