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Denaturing gradient gel electrophoresis gel expansion (DGGEGE) – An attempt to resolve the limitations of co-migration in the DGGE of complex polymicrobial communities

Gavin P. Gafan, David A. Spratt
DOI: http://dx.doi.org/10.1016/j.femsle.2005.09.048 303-307 First published online: 1 December 2005


Recent molecular approaches for the study of microbial communities such as PCR-cloning have enabled the detection and identification of as-yet-unculturable taxa. Cloning and sequencing of multiple samples is extremely laborious and expensive to perform thoroughly due to the large diversity involved. For this purpose, techniques such as denaturing gradient gel electrophoresis (DGGE) may be better suited. There is increasing evidence suggesting that DGGE of complex polymicrobial communities may be limited by co-migration of different sequences. In this study, we attempt to address this limitation by excising individual bands and running them through a shorter denaturant gradient, a process we have termed “denaturing gradient gel electrophoresis gel expansion” (DGGEGE).

  • 16S rDNA
  • Co-migration
  • Banding patterns
  • DGGE
  • Equivalent bands
  • Fingerprint analysis, OTU

1 Introduction

Denaturing gradient gel electrophoresis (DGGE) is a recent fingerprinting technique in which PCR-amplified DNA fragments are separated according to their sequence information [1]. This technique has been applied previously in environmental microbiology [24], food microbiology [5,6] and in the analysis of microbial communities inhabiting the human body [710]. Prior to DGGE, community studies of environmental samples had been achieved mainly by culture or via a PCR-cloning approach [1114]. Cloning and sequencing of multiple samples is an extremely laborious and expensive task to perform thoroughly due to the large diversity involved [12,15]. For this purpose, genetic fingerprinting techniques, such as DGGE, may be better suited. The main assumptions of DGGE when applied to the analysis of bacterial communities are that double stranded DNA molecules of the same length, but differing in base-pair sequence, can be partially separated as they migrate down a polyacrylamide gel containing a linearly increasing gradient of denaturants [16]. Theoretically, each DGGE band corresponds to a single operational taxonomic unit (OTU), where the total banding pattern is reflective of a community's species richness and diversity [17]. Earlier workers have excised and directly PCR-amplified and sequenced [18,19] or PCR-cloned and sequenced DGGE bands to successfully identify the taxonomic units of interest [20,21]. Conversely, recent investigators [2225] have reported that band excision and sequencing of DGGE bands might not provide unequivocal identifications as a result of the co-migration of DNA fragments from different taxa to the same positions within DGGE gels. The aim of this work is to attempt to minimise the limitation of co-migration in DGGE by running excised fragments through a shorter denaturing gradient, a process we have termed “denaturing gradient gel electrophoresis gel expansion” (DGGEGE).

2 Materials and methods

2.1 DGGE profiling

Dental plaque was sampled from seven pre-pubertal children (aged between 5 and 9 years) from the buccal and lingual gingival crevice of either the lower left or lower right first permanent molar tooth using a sterile toothpick. DNA was extracted from these samples using the Puregene™ DNA isolation Kit for yeast and Gram-positive bacteria (Gentra Systems, Minneapolis, USA). These DNA extracts were used as templates for a PCR reaction where the V2–V3 region of the 16S rRNA gene, corresponding to position 339–539 of Escherichia coli, was partially amplified using the primers (Genosys, Cambridgeshire, UK) F357GC (5′-CGCCCGCCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCCCCTACGGGAGGC-AGCAG-3′), which contains a GC-rich clamp, and R518 (5′-ATTACCGCGGCTGC-TGG-3′). The final volume of each PCR reaction mixture was 50 μl. The amplification reaction mixture contained 10× PCR buffer, 2.5 mM MgCl2, 0.2 μM of both primers F357GC and R518 and 5 U of Taq polymerase (Bioloine, London, UK). All dNTP's (Promega, Southampton, UK) were used at a final concentration of 0.2 mM. The cycling parameters for a touchdown PCR were carried out on a Primus thermal cycler (MWG Biotech, Milton Keynes, UK) and were as follows. After pre-incubation at 94 °C for 5 min, 30 cycles were performed at 94 °C for 1 min, TA for 1 min and 72 °C for 1 min. The TA decreased step-wise by 1 °C every 2 cycles from 65 °C in the first cycle to 56 °C in the 20th cycle. The TA for the last 10 cycles was 55 °C. Cycling was followed by 5 min incubation at 72 °C [21].

A parallel DGGE gel containing 10% (w/v) polyacrylamide (37.5:1 acrylamide:bisacrylamide) was cast using a D-code system (Bio-Rad laboratories Inc., Hercules, CA, USA). Each gel contained a linear gradient of the denaturants urea and formamide, increasing from 40% at the top of the gel to 80% at the bottom (with 100% denaturants corresponding to 7 M urea and 40% (v/v) deionised formamide). Gels were run at 35 V for 21 h (735 V h) at a constant temperature of 60 °C in 7 l of 1× TAE buffer. Gels were stained for 1 h in 1× TAE containing SYBR Green Nucleic Acid Gel Stain (10-4 dilution) (Molecular Probes, PoortGebouw, Netherlands) and photographed under a UV light transilluminator (AlphaImager, San Leandro, USA).

A single DGGE band that was present in all seven DGGE profiles was chosen (Fig. 1). These bands were checked as to whether they were equivalent to one another by using the EquiBands applet [26]. These equivalent bands were excised and the DNA fragments were then eluted in 20 μl of molecular grade water at 4 °C for 24 h [18]. All excised DGGE fragments were electrophoresed through a second, identical DGGE gel, in order to verify whether single DGGE bands had been excised.

Figure 1

DGGE profiles from dental plaque sampled from seven subjects. The ‘band of choice’ excised for DGGEGE is demarked by arrows.

2.2 Calculating DGGEGE denaturant gradient range

An image of the whole DGGE gel was captured using a UV light transilluminator (AlphaImager) and camera. The distance of electrophoretic migration for each band of interest within the DGGE gel was measured using the ruler option in the Adobe Photoshop 6.0 software package (San Jose, CA). The distance from the loading wells to the bottom of the gel was also measured. The denaturant concentration at which the band of interest migrated to could then be calculated using both of these distances together with the final denaturant concentration of 80%. DGGEGE gels were prepared with a denaturing gradient 2% above and below this calculated value.

2.3 DGGEGE profiling

Parallel DGGEGE gels containing 10% (w/v) polyacrylamide (37.5:1 acrylamide:bisacrylamide) were cast using a DCode system (Biorad laboratories Inc., Hercules, CA, USA). The gels contained a linear gradient of the denaturants urea and formamide, increasing from 50% at the top of the gel to 55% at the bottom (with 100% denaturants corresponding to 7 M urea and 40% (v/v) deionised formamide). PCR products (30 μl) and loading buffer (10 μl) were loaded onto the gels and run at 35 V for 21 h (735 V h) at a constant temperature of 60 °C in 7 l of 1× TAE buffer. Gels were stained for 1 h in 1× TAE containing SYBR Green Nucleic Acid Gel Stain (10-4 dilution) (Molecular Probes) and photographed under a UV light transilluminator (AlphaImager). This experiment was repeated to check whether DGGEGE produced reproducible fingerprints.

2.4 DGGEGE band excision, PCR-cloning and sequencing

DGGEGE bands were excised and the DNA fragments eluted. These fragments were amplified by a touchdown PCR using the primers 357F (no GC clamp) and 518R. The 3 equivalent DGGEGE fragments were PCR-cloned using the TOPO TA cloning kit (Invitrogen, Paisley, UK) and individual clones were PCR-sequenced. PCR products were sequenced using BigDyeTM terminator cycle sequencing version 2.0 ready reaction sequencing kit according to the manufacturer's instructions (Applied Biosystems) and analysed using a 310 Genetic Analyzer (AB biosystems). The primer 357F (0.1 μM) was used in the sequencing reaction.

3 Results and discussion

3.1 DGGE profile and band excision

Distinct DGGE profiles were observed for all seven samples (Fig. 1); the bands denoted by the arrow, ‘bands of choice’ were all shown to be equivalent (divergence of optimum <3) by using the EquiBands applet and were thus excised from all seven lanes. These specific bands were observed to stop migrating at a denaturant concentration of approximately 52–53%. The electrophoresis of these excised bands in a second identical DGGE gel demonstrated single bands that migrated to the same position, thus precluding the possibility that other DGGE bands had also been inadvertently excised.

3.2 DGGEGE profiling

PCR-amplified products from the excised and eluted bands were electrophoresed through a very short denaturant gradient (50–55%). The single bands of choice were expanded into mixed profiles (Fig. 2) with an average of 5.4 bands per lane (range of 4–8 DGGEGE bands). This demonstrated that co-migration of different sequences to the same electrophoretic position does take place in DGGE of dental plaque microbial communities. Separation was reproducibly shown to occur through a 50.6%–51.7% gradient of denaturants (Fig. 2). The purpose of this work was to develop a technique that would overcome the limitations imposed on DGGE by co-migration. The principle of DGGEGE is based on resolving these ‘co-migrating taxonomic units’ (CTUs) into individual OTUs. It would therefore be possible, albeit laborious, to excise every DGGE band from a single lane and then attempt to resolve each individual DGGE band into its constituent CTUs by DGGEGE. By aligning each DGGEGE profile accordingly, we hoped to generate a fingerprint that truly represented the dental plaque microbiota.

Figure 2

Duplicate DGGEGE profiles obtained from the single band of choice from seven different subjects. Gels A and B demonstrate duplicate expanded profiles. Lanes 1 and 8; lanes 2 and 9; lanes 3 and 10; lanes 4 and 11; lanes 5 and 12; lanes 6 and 13; lanes 7 and 14 demonstrate reproducible DGGEGE profiles form the bands of choice excised for the original DGGE gel.

3.3 DGGEGE band excision, PCR-cloning and sequencing

A total of 38 bands were excised from the DGGEGE gel (Fig. 3), direct PCR-sequencing of these bands provided sequence data that showed that the DGGEGE bands were mixed, i.e. contained multiple sequences. Three equivalent DGGEGE bands, 2, 7 and 12 (Fig. 2) were PCR-cloned and five random clones for each of the three bands were then sequenced. PCR-sequencing of the clones obtained from these 3 equiv. bands demonstrated that these bands were indeed mixed with several co-migrating taxa (Table 1). F. nucleatum and various streptococci were identified from all three bands along with 2 or 3 other taxa. Gram-positive and Gram-negative taxa were represented in all three bands. It may be assumed that the other 38 DGGEGE bands will probably also contain multiple co-migrating sequences. This demonstrates that, as in DGGE, DGGEGE bands appeared to be comprised of mixed co-migrating sequences belonging to different species of oral bacteria. Thus, DGGEGE could not resolve CTUs from equivalent DGGE profiles into individual OTUs. Perhaps when assessing extremely complex whole microbial communities, for example, those that exist in the oral cavity a DGGE approach is inappropriate and the use of a PCR-cloning approach is called for.

Figure 3

DGGEGE bands excised for sequencing; 38 bands were excised in total.

View this table:
Table 1

Taxa identified from the 3 equiv. excised DGGEGE bands 2, 7 and 12 (Fig. 3)

Band 2Band 7Band 12
Fusobacterium nucleatumFusobacterium nucleatumFusobacterium nucleatum
Gemella haemolysinsPeptostreptococcus microsHaemophilus parahaemophilus
Leptotrichia spp.Porphyromonas catoniaePorphyromonas catoniae
Neisseria flavescensPrevotella shahiiStreptococcus mitis
Streptococcus cristatusStreptococcus cristatusStreptococcus sanguinis


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