Akira Ichikawa and Seiji Igarashi
Tochigi Cancer Center, Tochigi, Japan
Denaturing gradient gel electrophoresis (DGGE) is one of the most consistently used mutation scanning methods. It has evolved enormously over the last decade to be widely used particularly in gene diagnostic laboratories. A practical method on this topic has been reported recently.1 The more frequently used mutation screening methods are DGGE and Single-Strand Conformational Polymorphism (SSCP). However, the sensitivity of DGGE is far higher (~95%) than that of SSCP, making it an attractive technique for screening unknown mutations.
The principle of DGGE is based on the fact that DNA duplexes melt at high temperature in the presence of a gradient of chemical denaturant. DNA melting is sequence specific and occurs in discrete segments called melting domains. Since melting involves breaking the hydrogen bonds that hold the base pair together, a G-C rich region melts at a higher temperature than an A-T rich region.
A PCR* product begins to melt from the region which has the lowest melting point (lowest Tm) first when it is exposed to chemical denaturant and high temperature. If there are point mutations in the PCR product, the melting point will change, and thus its mobility on a polyacrylamide gel will be different from the mobility of the wild type DNA. In other words, the mobility of the DNA fragment-containing mutation in the gel will be different from that of the wild type. In this paper we describe the use of DGGE to screen p53 mutations in breast cancer.
One hundred and two genomic DNA sam ples obtained from breast cancers were examined from exon 5, 6, 7 and 8 of the p53 gene. They were amplified by PCR with a pair of primers which contains a 40 bp GC clamp in one of them.2 All products were analyzed both in perpendicular gradient gels and parallel gradient gels.
PERPENDICULAR GRADIENT GEL ANALYSIS
A 7.5 x 10 cm, 1 mm thick, 25% to 65% denatured gradient gel was made using 6% aclylamide/bis (37.5:1) in 1x TAE buffer (50 mM Tris, 25 mM acetic acid, 1.25 mM EDTA). Four different PCR products each from exon 5, 6, 7 and 8 were mixed in a 25 l volume and the total sample volume made up to 100 l. We added 100 ml 2x gel loading dye (70% glycerol, 0.05% bromophenol blue, 0.05% xylene cyanol, 2 mM EDTA) to these samples, and electrophoresed them on the DCode system at 130 V for 2.5 hours at 56 C. After electrophoresis, the gels were stained in a 1:10,000 dilution of SYBR Green I (Molecular Probes) in 1x TAE buffer for 45 minutes and destained in 1x TAE buffer for 45 minutes. The gels were imaged under UV transillumination.
PARALLEL GRADIENT GEL ANALYSIS
A 16 x 16 cm, 1 mm thick, 45% to 60% denatured gradient gel was made using 6% aclylamide/bis (37.5:1) in 1xTAE buffer. Five ml of PCR products (approximately 200300 ng) was mixed with 5 l 2x gel loading dye and electrophoresed on the DCode system at 150 V for 3.5 hours at 60 C. The post-run analysis was the same as perpendicular gradient gel analysis.
Results and Discussion
The optimum concentration of denaturant to be used for parallel gradient gels was determined from the perpendicular gradient gel analysis. All exons separated and had a melting transiti on using a 25 to 65% gradient. Therefore a gradient of 45 to 60% was used for parallel gel analysis.
DNA from 34 cases was found to have a mutation on perpendicular gels and in 33 cases on parallel gels. The sample missed showed a mutation of exon 7 on perpendicular gels but not on parallel gels. A possible explanation for this could be that the parallel gel gradient of 45 to 60% was too wide for that particular sample. A narrow gradient may be required to identify the mutation of exon 7 on parallel gels.
We chose to use parallel gradient method because it provides high throughput analysis. The detection of mutation was unclear in some cases where the rate of mutated allele was low or the genomic DNA was extracted from paraffin embedded materials. In such cases we used SYBR Green I instead of ethidium bromide to stain the gels. This improved sensitivity and enabled us to see the bands clearly.
These experiments indicate that DGGE is a powerful technique to screen mutations. The DCode system is easy-to-use and reliable, two important factors when scanning numerous mutations.
1. Foddel, R. and Losekoot, M., Human Mutation, 3, 8394 (1994).
2. Anderson, T. I. and Boreson, A-L., Diag. Mol. Patho., 4, 203211 (1995).
The Polymerase Chain Reaction (PCR) process is covered by patents owned by Hoffmann-LaRoche. Use of the PCR process requires a license.
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