Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer

Cecilia Gelfi, Agnese Viganó, Mario Curcio, Pier Giorgio Righetti, Sabina Carla Righetti, Elisabetta Corna, Franco Zunino

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)785-791
Number of pages7
JournalElectrophoresis
Volume21
Issue number4
DOIs
Publication statusPublished - 2000

Fingerprint

Capillary Electrophoresis
Electrophoresis
Polymorphism
Conformations
Buffers
Gels
Ions
Temperature
Morpholinos
Tromethamine
Polymerase chain reaction
Ethylenediaminetetraacetic acid
Reaction products
Acids
Electric potential
Polymerase Chain Reaction
Mutation

Keywords

  • p53
  • Point mutation
  • Single-strand conformation polymorphism

ASJC Scopus subject areas

  • Clinical Biochemistry

Cite this

Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer. / Gelfi, Cecilia; Viganó, Agnese; Curcio, Mario; Righetti, Pier Giorgio; Righetti, Sabina Carla; Corna, Elisabetta; Zunino, Franco.

In: Electrophoresis, Vol. 21, No. 4, 2000, p. 785-791.

Research output: Contribution to journalArticle

Gelfi, Cecilia ; Viganó, Agnese ; Curcio, Mario ; Righetti, Pier Giorgio ; Righetti, Sabina Carla ; Corna, Elisabetta ; Zunino, Franco. / Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer. In: Electrophoresis. 2000 ; Vol. 21, No. 4. pp. 785-791.
@article{4b0250c623a8458c97a48e54435e1f6b,
title = "Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer",
abstract = "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.",
keywords = "p53, Point mutation, Single-strand conformation polymorphism",
author = "Cecilia Gelfi and Agnese Vigan{\'o} and Mario Curcio and Righetti, {Pier Giorgio} and Righetti, {Sabina Carla} and Elisabetta Corna and Franco Zunino",
year = "2000",
doi = "10.1002/(SICI)1522-2683(20000301)21:4<785::AID-ELPS785>3.0.CO;2-E",
language = "English",
volume = "21",
pages = "785--791",
journal = "Electrophoresis",
issn = "0173-0835",
publisher = "Wiley-VCH Verlag",
number = "4",

}

TY - JOUR

T1 - Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer

AU - Gelfi, Cecilia

AU - Viganó, Agnese

AU - Curcio, Mario

AU - Righetti, Pier Giorgio

AU - Righetti, Sabina Carla

AU - Corna, Elisabetta

AU - Zunino, Franco

PY - 2000

Y1 - 2000

N2 - 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.

AB - 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.

KW - p53

KW - Point mutation

KW - Single-strand conformation polymorphism

UR - http://www.scopus.com/inward/record.url?scp=0034061152&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034061152&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1522-2683(20000301)21:4<785::AID-ELPS785>3.0.CO;2-E

DO - 10.1002/(SICI)1522-2683(20000301)21:4<785::AID-ELPS785>3.0.CO;2-E

M3 - Article

VL - 21

SP - 785

EP - 791

JO - Electrophoresis

JF - Electrophoresis

SN - 0173-0835

IS - 4

ER -