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Clinical Chemistry 45: 35-40, 1999;
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(Clinical Chemistry. 1999;45:35-40.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Validation of Double Gradient Denaturing Gradient Gel Electrophoresis through Multigenic Retrospective Analysis

Laura Cremonesi1,a, Paola Carrera1, Antonella Fumagalli1, Sabrina Lucchiari1, Elena Cardillo1, Maurizio Ferrari1, Sabina Carla Righetti2, Franco Zunino2, Pier Giorgio Righetti3 and Cecilia Gelfi4

1 Instituto di Ricovero e Cura a Caraterre Scientifico, O San Raffaele, Unità di Genetica e Diagnostica Molecolare, Via Olgettina 60, 20132 Milan, Italy.

2 Istituto Nazionale Tumori, Oncologia Sperimentale B, 20153 Milan, Italy.

3 Università di Verona, Facoltà di Scienze, 37134 Verona, Italy.

4 Instituto Technologie Biomediche Avanzate, Consiglio Nazionale delle Ricerche, Segrate (Milano), Italy.
a Author for correspondence. Fax 392 2643 4767; e-mail cremonisi.laura{at}hsr.it.


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Among established techniques for the identification of either known or new mutations, denaturing gradient gel electrophoresis (DGGE) is one of the most effective. However, conventional DGGE is affected by major drawbacks that limit its routine application: the different denaturant gradient ranges and migration times required for different DNA fragments. We developed a modified version of DGGE for high-throughput mutational analysis, double gradient DGGE (DG-DGGE), by superimposing a porous gradient over the denaturant gradient, which maintains the zone-sharpening effect even during lengthy analyses. Because of this innovation, DG-DGGE achieves the double goals of retaining full effectiveness in the detection of mutations while allowing identical run time conditions for all fragments analyzed. Here we use retrospective analysis of a large number of well-characterized mutations and polymorphisms, spanning all predicted melting domains and the whole genomic sequence of three different genes—the cystic fibrosis transmembrane conductance regulator (CFTR), the ß-globin, and the p53 genes—to demonstrate that DG-DGGE may be applied to the rapid scanning of any sequence variation.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The increasing demand for mutation detection in disease genes, either known or presumed, can be answered by automated sequencing using fluorescent dyes (1); however, only a few laboratories are equipped for the broad application of this costly and labor-intensive strategy. As alternatives to sequencing methods, which determine the exact nature and location of each base along a DNA region, various mutation-scanning procedures have been developed. These methods, which rely on the recognition of a sequence variation between presumptive mutant and wild-type (wt)1 DNA on the basis of an altered electrophoretic migration pattern, provide a simple means for determining whether a given DNA sample harbors a mutation in a particular gene.

The most established scanning procedures are single-strand conformational polymorphism (SSCP) (2), denaturing gradient gel electrophoresis (DGGE) (3)(4), chemical cleavage of mismatch (5), RNase cleavage (6), and heteroduplex analysis (7). Among these methods, SSCP, DGGE, and heteroduplex analysis are the most widely used because of their simplicity and/or nontoxicity.

In recent years, no striking novel methodologies have been developed; however, substantial improvements have been made to the established protocols, aimed at improving sensitivity, simplicity, and economy, all important factors in view of large-scale clinical applicability.

We focused on DGGE because this method, when optimized, displays the highest mutation detection rate (close to 100%) (8) compared with SSCP and heteroduplex analysis. Additional advantages of this methodology are the possibility of optimizing the analysis by computer simulation [based on the Melt87 and SQHTX programs of Lerman and Silverstein (3)] and a nonradioactive protocol.

DGGE consists of coamplification of the wt and presumptive mutant DNAs to obtain, before analysis, a family of fragments: two hetero- and two homoduplexes. Separation among the various members of the family will not occur when electrophoresis is performed in the absence of denaturants; the minute differences in the radius of gyration (Rg), caused by the mismatch between the two hetero- and homoduplexes will lead, at best, only to band broadening. However, if the same set of duplexes is run against a gradient of denaturants (either chemical or thermal), each duplex at some point during migration pass through an isoperichoric milieu in which the microenvironment will exactly match the melting temperature of the lowest melting domain of the duplex. This domain will suddenly be destabilized, and transition from an orderly helix to a partially unwound DNA molecule will take place; the markedly increased frictional drag between the partially unwound DNA, the gel matrix, and solvent molecules will retard DNA mobility. The small, but not negligible, differences in the Tm values of the various duplexes typically will allow a spectrum of four bands to be resolved, indicating the presence of a mutation along a DNA filament. Small shifts in the Tm of the corresponding melting domain may be caused by a conservative transversion (AT->TA, GC->CG) or by mutations at the very end of cooperative melting domains, whereas switches of G:C pairs to A:T and vice versa, small deletions or insertions, or multiple mutations usually lead to larger shifts in Tm.

For the technique to be successful, different conditions must be found and optimized because the slopes of the denaturant gradient and running times vary for every DNA region to be analyzed; in addition, the long running time needed to resolve hetero- and homoduplexes often produces smears, affecting detectability. To greatly improve the reliability of the system and to make it more suitable for routine use, we added a porosity gradient over the denaturing gradient [double gradient DGGE (DG-DGGE)]. The porosity gradient suppresses band broadening even in lengthy electrophoretic runs, thus allowing uniform run times and conditions for all fragments while maintaining the zone-sharpening effect. Previously, we demonstrated the enhanced power of DG-DGGE compared with conventional DGGE in detecting heteroduplexes that were almost indistinguishable from the ethidium bromide background and in resolving even homoduplexes that appeared as single bands (9). In the present study, we applied DG-DGGE to every possible gradient slope through the retrospective analysis of a large sample of previously identified mutations and polymorphisms, including several conservative transversions, located in the genes that code for cystic fibrosis transmembrane conductance regulator (CFTR), ß-globin, and p53.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
dna samples containing known mutations
The retrospective analysis was carried out on DNA samples carrying 98 mutations and polymorphisms, among which 49 were located in the CFTR gene (Table 1 ) (10), 27 were located in the ß-globin gene (Table 2 ) (11), and 22 were located in the p53 gene (Table 3 ) (12)(13)(14)(15). Mutations and polymorphisms had been identified previously by other techniques, such as either DGGE or SSCP coupled with direct sequencing, dot-blotting, and the amplification refractory mutation system (16).


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  		Table 1. Mutations and polymorphisms analyzed in the CFTR gene.


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  		Table 2. Mutations and polymorphisms analyzed in the ß-globin gene.


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  		Table 3. Mutations analyzed in the p53 gene.

The mutations and polymorphisms included 83 single nucleotide substitutions, 12 deletions, 2 insertions, and a composed deletion + substitution mutation. A total of 16 conservative transversions (11 C/G and 5 A/T) were examined (Tables 1–3Up Up Up ). These sequence alterations spanned 20 genomic regions (20 exons, including their flanking splice site sequences) of the CFTR gene, 6 regions (the promoter, 2 exons, 2 introns, and the 3' untranslated region) of the ß-globin gene, and 3 regions (3 exons, including intron/exon boundaries) of the p53 gene.

dg-dgge conditions
The group of mutations considered in this study spanned every possible predicted melting domain, as analyzed by the most commonly used ranges of denaturant, including low-melting (10–60%), intermediate low-melting (20–70%), intermediate high-melting (30–80%), and high-melting domains (40–90%). GC-clamp sequences, primer sequences, PCR conditions, and denaturant gradients for mutation analysis in the CFTR (17), ß-globin (18)(19)(20), and p53 (21)(22) genes were as described previously. Heteroduplexes were generated at the end of each PCR session (5 min at 94 °C, 1 h at 56 °C). PCR-amplified fragment lengths ranged from 168 to 516 bp, with most of the fragments being 200–400 bp long. A 15-µL aliquot of each amplified DNA sample was subjected to electrophoresis in a 6.5–12% T polyacrylamide gel containing a linear porosity gradient (T indicates the total percentage of monomers, acrylamide, and bisacrylamide, with a 39:1 ratio for the acrylamide and bisacrylamide) in Tris-EDTA-acetate buffer (40 mmol/L Tris, 20 mmol/L NaOH, and 1 mmol/L EDTA titrated to pH 7.6 with acetic acid) and in the presence of the same linear gradient of denaturants, urea and formamide, as used for standard DGGE (7 mol/L urea and 400 mL/L formamide represent the 100% denaturant). For all samples, however, a single set of running conditions was adopted: 75 V for 15 h. The gel slabs were 0.75 mm thick, 15 cm wide, and 16 cm long. At the end of the electrophoresis, the gels were stained in ethidium bromide.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
retrospective mutational analysis by dg-dgge
To demonstrate the large applicability of the DG-DGGE technique, a retrospective analysis of mutations and polymorphisms located in three different genes was performed. These genes were chosen for this study on the basis of different considerations. The first criterion adopted was the variety of the genes in location, function, and genome organization. As shown in Tables 1–3Up Up Up , the sequence alterations selected for this study were scattered among numerous sites within the three genes examined. The second criterion was to include genes containing melting domains representative of all possible denaturant gradients, such as low-, intermediate low-, intermediate high-, and high melters. As shown in Tables 1–3Up Up Up , whereas sequences amplified from the CFTR gene coding region are mostly low- and intermediate low melters, amplicons from both the ß-globin and the p53 genes belong to the intermediate high- and high melter categories. The final criterion was to include mutations representative of all situations, such as deletions, insertions, and nucleotide substitutions, comprising several conservative transversions. These last mutations in particular are the most difficult sequence alterations to detect, because they do not alter the overall base composition of the DNA fragment and very often their detectability is based solely on the presence of the heteroduplex species.

All of the 98 mutations and polymorphisms analyzed by DG-DGGE produced an anomalous electrophoretic pattern displaying sharp zones for both homo- and heteroduplexes in the DNA fragment where the mutation was expected to occur, confirming the full effectiveness in detecting mutations under the optimized conditions already reported for conventional DGGE (8), even on a heterogeneous fragment population based on fragment length.

Figs. 1 -4 showsome examples of DG-DGGE analysis on different mutations, grouped according both to the gradient of denaturant used and to the gene examined. In particular, Fig. 3 shows the analysis of five conservative transversions located in different regions of the ß-globin gene. Fig. 4 shows the electrophoretic pattern corresponding to samples derived from biopsies of different ovarian tumors. PCR-amplified samples from ovarian tumor tissues may show an unbalanced ratio between the wt homoduplex (wt/wt) and the mutated homoduplex (m/m) zones, as shown in lanes 4–6. The wt homoduplex is generated from endogenous genomic material belonging to the nondiseased cell population still present in the tumor mass (21).



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  		Figure 1. Detection of different mutations in intron 2 and the UTR3' region of the ß-globin gene by DG-DGGE.

Gel contains a porosity gradient of 6.5–12% T and a denaturant gradient of 10–60%. Separation of PCR fragments 5 (from IVS2-562 to +1475) and 7 (from IVS2-768 to +1666) is shown. Lane 1, fragment 5, wt; lane 2, fragment 5, IVS2-745 C->G/wt; lane 3, fragment 7 wt'; lane 4, fragment 7, polyA site A->G in UTR3'/wt; lane 5, fragment 5, IVS2-666T->C/wt.



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  		Figure 3. Detection of different conservative transversions in the ß-globin gene by DG-DGGE.

Gel contains a porosity gradient of 6.5–12% T and a denaturant gradient of 30–80%. Separation of four PCR fragments is shown: fragment 1 (from -238 to -37); fragment 2 (from -123 to codon 21); fragment 3 (from -123 to IVS1-119); and fragment 4 (from IVS1-29 to IVS2-30). Lane 1, fragment 1, -87 C->G/wt; lane 2, fragment 1, wt; lane 3, fragment 2, -30 T->A/wt; lane 4, fragment 2, codon 6 A->T (HbS)/wt;lane 5, fragment 2, wt; lane 6, fragment 3, codon 30 G->C/wt; lane 7, fragment 3, wt; lane 8, fragment 4, IVS1-130 G->C/wt; lane 9, fragment 4, wt.



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  		Figure 4. Detection of different mutations in exon 7 of the p53 gene by DG-DGGE.

Gel contains a porosity gradient of 6.5–12% T and a denaturant gradient of 40–90%. Lane 1, codon 250 C->T/wt; lane 2, codon 237 G->A/wt; lane 3, codon 237 G->T/wt; lane 4, codon 245 G->A/wt; lane 5, codon 242 G->T/wt; lane 6, codon 236 A->G/wt; lane 7, wt.

advantages of dg-dgge
Among mutation screening methods, DGGE is not yet the most widely used, although it fits most of the criteria for an optimal technique, being accurate, fast, inexpensive, and easy to perform, while avoiding the use of radioactive material. The use of DGGE is limited mostly because the denaturant slope and running times must first be optimized for the different DNA fragments examined, which requires more time than other scanning techniques. Actually, although primer location, running times, and denaturant slopes can be predicted for every DNA fragment by computer modeling using algorithms developed by Lerman and Silverstein (3), conditions still need to be optimized empirically. Because the migration time needed for resolving the homoduplexes frequently exceeds that needed for separating the heteroduplexes, the latter often produce less-sharp or even blurred bands, scarcely distinguishable from the background fluorescence after ethidium bromide staining.

The masking of heteroduplexes can have a major impact on the detection of conservative transversions because homoduplex species often comigrate with the heteroduplexes in these samples, which leads to false-negative results.

Moreover, the gel-to-gel variability that occurs when gels are cast manually can also affect resolution. In addition, we observed that the purity of reagents and the quality of primers are crucial for reproducibility of the technique and can seriously affect the routine application of the method.

In an attempt to address this problem, we concluded that a porosity gradient was the simplest way to minimize band broadening during analysis and to render the technique more robust. For this purpose, we developed DG-DGGE, which superimposes a porous gradient over the denaturant gradient, allowing greatly improved band resolution even during lengthy analyses. This allows the use of a single set of running conditions for the analysis of all DNA regions sharing the same melting profile. In particular, for the ß-globin gene, the use of only two denaturant gradients (10–60% and 30–80%) allows analysis of 27 very common mutations. Similarly, in the CFTR gene, most exons share the same melting profiles and can be examined with the use of two ranges of denaturant (10–60% and 20–70%). Because mutations in this gene span the entire coding region, this greatly reduces the time needed to scan the entire gene in each sample.

Moreover, in laboratories where several disease genes are studied routinely, different DNA fragments, even from different genes, can be analyzed in the same gel. Furthermore, the improved zone resolution obtained by DG-DGGE does not require any additional specialized equipment because it uses the same two-vessel gradient mixer used for pouring the standard DGGE slab.

In addition, in samples where the ratio between the two homoduplexes is far from one, as in samples containing unequal amounts of wt and mutated fragments (e.g., in tumor biopsies), it is possible to perform densitometry of all zones and assess their relative ratio because of the very sharp zones generated by DG-DGGE. This allows the determination of the percentage of cells in a population that have developed a given mutation and may have relevant implications for the pathogenesis, diagnosis, prognosis, and therapy of human cancer.

The majority of scanning procedures yield no information regarding the location or nature of the mutation within a given DNA fragment, and these methods are always coupled with direct sequencing for precise characterization. This deficiency is partially overcome by the present method because the enhanced resolution of both homo- and heteroduplexes obtained by DG-DGGE improves the diagnostic power of the technique, allowing better assignment of a single banding pattern to a specific mutation. The method could be used to screen for a panel of mutations known to be predominant in a given population. In this case, the mutation identified on the basis of the altered migration pattern could be confirmed by simple specific tests such as the amplification refractory mutation system, restriction digestion, or dot-blotting, while avoiding sequencing.

On the basis of these considerations and also taking into account that DG-DGGE is an optimized version of conventional DGGE, which is known to display the highest detection rate with respect to any other scanning technique, DG-DGGE could become the method of choice for identifying mutations at multiple potential sites within any segment of DNA for which the wt sequence is known.



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  		Figure 2. Detection of different mutations in exon 17b of the CFTR gene by DG-DGGE.

Gel contains a porosity gradient of 6.5–12% T and a denaturant gradient of 20–70%. Lane 1, F1052V (T->G at 3286)/wt; lane 2, R1066C (C->T at 3328)/wt; lane 3, R1066H (G->A at 3329)/wt; lane 4, wt.


   Acknowledgments
 
This work was supported in part by Associazione Italiana per la Ricerca sul Cancro, Telethon E.555 (P.G. Righetti), the Ministero della Sanità, Progetto Fibrosi Cistica, and Bio-Rad Laboratories (M. Ferrari).


   Footnotes
 
1 Nonstandard abbreviations: wt, wild-type; SSCP, single-strand conformational polymorphism; DGGE, denaturing gradient gel electrophoresis; DG, double gradient; and CFTR, cystic fibrosis transmembrane conductance regulator.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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