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]

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Material and Methods

Dynabeads M-280 Streptavidin were purchased from Dynal AS (Oslo, Norway). Ultrafree-MC filters were from Millipore (Bedford, MA). Methylcellulose (high viscosity) and other chemicals were from Fluka (Buchs, Switzerland).

PCR amplifications were performed as follows (6): 500 ng of genomic DNA was added to a standard (100µl) PCR mix containing 2.5 U Taq polymerase (Finnzyme), 1.5 mM MgCl₂, 200 µM dNTPs and 0.2 µM primers (Ap1-bio and R94). The mixture was cycled 30 times at 94°C for 1 minute, 55°C for 1 minute and 72°C for 2 minutes. The PCR product (100 µl) was incubated with 400 µg of prewashed Dynabeads for 15 minutes. The supernatant was removed using a magnetic device and the beads were washed twice with Tris buffer, pH 8.0 and treated for 10 minutes with NaOH (0.1 M) at room temperature to denature the amplified product. The alkaline supernatant containing the non-biotinylated strand was transferred to a clean tube and neutralized with HCl, 0.1 M. Before loading on CE the samples were desalted with Ultrafree-MC filters (cut off 30kD). The final volume was about 50 µl.

CE was performed on a BioFocus 3000 system (BioRad, Hercules, CA) (in the reversed polarity mode) using coated fused-silica capillary (50 cm x 50 cm or 36 cm x 50 µm, BioRad). Samples were introduced into the capillary tube using electrokinetic injection (at 8 kV for 20 sec). Separation within the coated capillary tube was performed under a constant voltage. The CE buffer consisted of 100 mM Tris, 100 mM boric acid, 2 mM EDTA and methylcellulose (high viscosity) at concentrations ranging from 0.1 to 0.6% (w/v). On-column detection was performed by UV adsorption at 260 nm, and the temperature of the capillary was set at 15°C. Solid phase fluorescent DNA sequencing was performed on positive samples following a published procedure (6).