To the Editor:
The continuing progress in the identification and characterization of genes that cause human genetic diseases has greatly increased the requests for a method that can rapidly identify mutations in these genes. The introduction of PCR has made it possible to rapidly obtain a relatively large amount of single-copy genomic DNA that can be used for subsequent analysis. Direct sequencing of amplified DNA, although a standard procedure in many laboratories, is still time-consuming and laborious. Consequently, different methods have been developed to pre-identify the region of the gene in which the mutation is located, which can subsequently be amplified and sequenced. We have chosen denaturing gradient gel electrophoresis (DGGE) of PCR-amplified material, by which fragments up to 800 bp in length can be screened for the presence of mutations. DGGE, as described originally (1), relies on the partial denaturation of a DNA duplex, which slows its movement in an increasing gradient of denaturant. If a mutation is present in the melted portion, the denaturing properties are changed and the duplex is arrested at a different point, signaling the mutation. This method has been modified so that it detects 90% or more of single base-pair substitutions (2). In standard DGGE methods, polyacrylamide gels are ethidium-bromide stained.
As far as we know from searching the literature and from personal communications, most laboratories using DGGE use ethidium bromide although it is a powerful mutagen and is not as sensitive as silver staining. When DGGE is used for screening large genes (e.g., CFTR, FVIII) and to screen >20 exons, it is convenient to rely on a method that is both sensitive and economical. Silver staining reduces the cost of testing by reducing the volume of the PCR mixture to 10 μL, improves the sensitivity, and provides a permanent record of results.
To illustrate the advantages of the combination of DGGE and silver staining, we performed DGGE of multiplex C for the CFTR gene under the conditions given by Fanen et al. (3). PCR was performed in a final volume of 10 μL, and a 1.5-μL aliquot was used for DGGE. The gel was silver-stained using the following protocol (Fig. 1⇓ ). After fixation in 150 mL of a solution of absolute ethanol (100 mL/L) and acetic acid (5 mL/L) for 10 min at room temperature with gentle shaking, staining was performed in 150 mL of a 1 g/L silver nitrate solution for 10 min at room temperature with gentle shaking. The gel was rinsed twice with distilled water to remove excess silver solution and was developed in a solution containing, per liter, 15g of NaOH, 0.1 g of NaBH4, and 0.48 g of formaldehyde. The gel was first washed in 150 mL of this solution (for ∼30 s) to precipitate the excess silver, and then was incubated in 150 mL of the same solution, with gentle shaking, until the bands were visualized (∼20 min). The stain was fixed with an aqueous solution of 7.5 g/L NaCO3 for 10 min at room temperature with gentle shaking. The gel was laid on a bench top and covered with Whatman 3MM paper. The Whatman paper was picked up and turned over, so that the gel was peeled off the bench top. The gel was covered with Saran Cling Wrap and stored at 4 °C (up to 2 weeks) or dried under reduced pressure for 1.5 h at 80 °C.
We believe this procedure is useful both for its economical cost and for its sensitivity.
This work was supported by a grant from the ICGEB (Grant CRP 47 YUG96-05). We thank Dr. Cecile Cazeneuve and ECCACF for obtaining primers for DGGE.
- © 2000 The American Association for Clinical Chemistry