Abstract
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°C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2°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.
| Original language | English |
|---|---|
| Pages (from-to) | 1517-1523 |
| Number of pages | 7 |
| Journal | Electrophoresis |
| Volume | 23 |
| Issue number | 10 |
| DOIs | |
| Publication status | Published - 2002 |
Fingerprint
Keywords
- p53
- Point mutations
- Single-strand conformation polymorphism
ASJC Scopus subject areas
- Clinical Biochemistry
Cite this
Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis. / Gelfi, Cecilia; Vigano, Agnese; De Palma, Sara; Righetti, Pier Giorgio; Righetti, Sabin Carla; Corna, Elisabetta; Zunino, Franco.
In: Electrophoresis, Vol. 23, No. 10, 2002, p. 1517-1523.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis
AU - Gelfi, Cecilia
AU - Vigano, Agnese
AU - De Palma, Sara
AU - Righetti, Pier Giorgio
AU - Righetti, Sabin Carla
AU - Corna, Elisabetta
AU - Zunino, Franco
PY - 2002
Y1 - 2002
N2 - 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°C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2°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.
AB - 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°C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2°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.
KW - p53
KW - Point mutations
KW - Single-strand conformation polymorphism
UR - http://www.scopus.com/inward/record.url?scp=0036072674&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0036072674&partnerID=8YFLogxK
U2 - 10.1002/1522-2683(200205)23:10<1517::AID-ELPS1517>3.0.CO;2-I
DO - 10.1002/1522-2683(200205)23:10<1517::AID-ELPS1517>3.0.CO;2-I
M3 - Article
VL - 23
SP - 1517
EP - 1523
JO - Electrophoresis
JF - Electrophoresis
SN - 0173-0835
IS - 10
ER -