Solid phase purification and SSCP analysis of amplified genomic DNA by capillary electrophoresis

Silvana Debernardi, Massimo Luzzana and Gianluca De Bellis

Consiglio Nazionale delle Ricerche, Istituto di Tecnologie Biomediche Avanzate, L.I.T.A., Via Fratelli Cervi 93, 20090 Segrate, ITALY
Email debellis@sun2.itba.mi.cnr.it
[Received 11/28/95; Accepted 12/25/95; On-line 1/1/96]

Introduction

Several methods have been proposed since the introduction of DNA amplification for the identification of mutations in genomic DNA. Due to its simplicity single stranded conformational polymorphism (SSCP) is the most frequently encountered in recent literature (1).

Magnetic solid phase DNA purification has gained wide popularity for either fluorescent or radioactive DNA sequencing (4,5).

Capillary electrophoresis permits high speed separation of biopolymers including double stranded and single stranded DNA (see 2 for a recent review). This high resolution analytical technique is very sensitive and reproducible and in addition allows automated instruments to quickly explore several analytical conditions to enhance the separation in specific samples. SSCP analysis in capillary electrophoresis has been already proposed for detection of the p53 mutation (3).

This paper presents a procedure to detect point mutations in amplified DNA by solid phase magnetic purification (4), single stranded DNA separation, SSCP analysis carried out on an automated CE apparatus and fluorescent automated solid phase DNA sequencing of the positive samples. The human ß globin gene was used as a test for this procedure.