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Single‐strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer
Author(s) -
Gelfi Cecilia,
Viganó Agnese,
Curcio Mario,
Righetti Pier Giorgio,
Righetti Sabina Carla,
Corna Elisabetta,
Zunino Franco
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/(sici)1522-2683(20000301)21:4<785::aid-elps785>3.0.co;2-e
Subject(s) - capillary electrophoresis , chemistry , tris , chromatography , analytical chemistry (journal) , hydroxymethyl , buffer (optical fiber) , electrophoresis , buffer solution , capillary action , ion , materials science , stereochemistry , biochemistry , organic chemistry , telecommunications , computer science , composite material
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 m M 2‐( N ‐morpholino)propanesulfonic acid (MES), 30 m M tris(hydroxymethyl)aminomethane (Tris), 1 m M 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.