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[DESCRIPTION]
Using DGGE, similar-sized PCR products that differ in nucleotide composition (sequence) can be separated in denaturing gradient gels. Denaturing gradient gels are created using acrylamide (structural material) solutions that contain different amounts of denaturants (urea and formamide), such that the highest concentration of denaturants is at the bottom of the gel and the lowest concentration is at the top. As dsDNA fragments differing in sequence migrate into the gel during electrophoresis, they encounter increasing concentrations of denaturants, and each fragment partially melts (i.e., double-stranded regions dissociate into single-stranded) at a different place in the gel depending on its sequence. Because the electrophoretic mobility of partially melted DNA fragments is greatly reduced compared to dsDNA, same-sized DNA fragments with different sequences focus at different positions in these gels. Because PCR with universal primers can be used to amplify related, but different, DNA sequences, and different sequences focus at different positions in a denaturing gradient gel, DGGE can be used to produce a unique banding pattern, or fingerprint, for each PCR product amplified from different microbial communities.

[GUIDELINES]
<strong>Equipment and reagents:</strong> <br /><br />* DGGE apparatus, e.g., Hoefer Scientific SE600, Bio-Rad DCode system (includes gradient former), Ingeny, CBS Scientific; <br /><br />* Peristaltic pump and gradient maker for casting gradient gels, e.g., SG Gradient Maker (GE Healthcare); <br /><br />* Power supply for electrophoresis systems; <br /><br />* Denaturing gel solutions: Solution A (for 250 mL of an 8% gel solution with 0% denaturant), 50 mL 40% acrylamide/bis stock solution (37:5:1 acrylamide:bis-acrylamide solution), 2.5 mL 50× TAE, adjust to 250 mL with sterile distilled water (sdH<sub>2</sub>O); and Solution B (for 250 mL of an 8% gel solution with 100% denaturant; i.e., 7 M urea and 40% formamide), 50 mL 40% acrylamide/bis stock solution (37:5:1 acrylamide:bis-acrylamide solution), 2.5 mL 50× TAE, 105 g urea, 100 mL deionized formamide, adjust to 250 mL with sdH<sub>2</sub>O; <br /><br />* 10% wt/vol ammonium persulfate solution in sdH<sub>2</sub>O (APS); this reagent should be prepared fresh each time it is used; <br /><br />* TEMED (N,N,N′,N′ -tetramethylenediamine); <br /><br />* 1× TAE running buffer (from 50× buffer stock solution: 20 mM Tris-acetate (pH 7.4), 10 mM sodium acetate, 0.5 mM EDTA); <br /><br />* 6× loading buffer (e.g., 60% glycerol, 10 mM Tris-HCl, pH 7.6, 60 mM EDTA, 0.03% bromophenol blue, 0.03% xylene cyanol FF);<br /><br />* Fluorescent DNA strain (e.g., SYBR Gold or SYBR II gel stain; Molecular Probes); <br /><br />* UV transilluminator and gel documentation system.<br /><br /><strong>Figures and Tables:<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bbsc2w6.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/bbrc2w6.jpg" data-ofn="fig1.jpg" /><br /><br />Fig. 1. </strong>Flowchart of methods involved in figerprinting aquatic virus communities.<strong><br /><br /><br /></strong><br /><strong>Table 1. Formulation for DGGE gels using 0% and 100% denaturing solutions.</strong><br /><br />
<table>
<tbody>
<tr>
<td>% denaturant of desired gel solution</td>
<td>Volume of solution A (0% denaturants)</td>
<td>Volume of solution B (100% denaturants)</td>
</tr>
<tr>
<td>0</td>
<td>14.50</td>
<td>0.00</td>
</tr>
<tr>
<td>15</td>
<td>12.32</td>
<td>2.18</td>
</tr>
<tr>
<td>20</td>
<td>11.60</td>
<td>2.90</td>
</tr>
<tr>
<td>25</td>
<td>10.87</td>
<td>3.63</td>
</tr>
<tr>
<td>30</td>
<td>10.15</td>
<td>4.35</td>
</tr>
<tr>
<td>40</td>
<td>8.70</td>
<td>5.80</td>
</tr>
<tr>
<td>45</td>
<td>7.97</td>
<td>6.53</td>
</tr>
<tr>
<td>50</td>
<td>7.25</td>
<td>7.25</td>
</tr>
<tr>
<td>55</td>
<td>6.54</td>
<td>7.96</td>
</tr>
<tr>
<td>60</td>
<td>5.80</td>
<td>8.70</td>
</tr>
<tr>
<td>65</td>
<td>5.07</td>
<td>9.43</td>
</tr>
<tr>
<td>70</td>
<td>4.35</td>
<td>10.15</td>
</tr>
</tbody>
</table>
<br /><strong><strong>Table 2. Denaturant amounts for various denaturing gel solutions.*<br /><br /></strong></strong>
<table>
<tbody>
<tr>
<td><strong>Denaturant </strong><br /><strong>concentration</strong></td>
<td style="text-align: center;"><br /><strong>10%</strong></td>
<td style="text-align: center;"><br /><strong>20%</strong></td>
<td style="text-align: center;"><br /><strong>30%</strong></td>
<td style="text-align: center;"><br /><strong>40%</strong></td>
<td style="text-align: center;"><br /><strong>50%</strong></td>
<td style="text-align: center;"><br /><strong>60%</strong></td>
<td style="text-align: center;"><br /><strong>70%</strong></td>
<td style="text-align: center;"><br /><strong>80%</strong></td>
<td style="text-align: center;"><br /><strong>90%</strong></td>
</tr>
<tr>
<td>Formamide, mL</td>
<td style="text-align: center;">4</td>
<td style="text-align: center;">8</td>
<td style="text-align: center;">12</td>
<td style="text-align: center;">16</td>
<td style="text-align: center;">20</td>
<td style="text-align: center;">24</td>
<td style="text-align: center;">28</td>
<td style="text-align: center;">32</td>
<td style="text-align: center;">36</td>
</tr>
<tr>
<td>Urea, g</td>
<td style="text-align: center;">4.2</td>
<td style="text-align: center;">8.4</td>
<td style="text-align: center;">12.6</td>
<td style="text-align: center;">16.8</td>
<td style="text-align: center;">21.0</td>
<td style="text-align: center;">25.2</td>
<td style="text-align: center;">29.4</td>
<td style="text-align: center;">33.6</td>
<td style="text-align: center;">37.8</td>
</tr>
</tbody>
</table>
<br />*For 8% acrylamide gels, add 20 mL of a 40% acrylamide/bis (37:5:1) solution, 2 mL 50x TAE, and bring the volue up to 100 mL using sdH<sub>2</sub>0<strong><br /><br />Analysis of PFGE and DGGE fingerprints:</strong><br /><br />DGGE and PFGE fingerprints can be analyzed using a variety of commercially available gel analysis software products (e.g., GelCompar II, Applied Maths; BioNumerics 5.1, Applied Maths; Quantity One, Bio-Rad). A common method to analyze DGGE/PFGE fingerprints involves creating a binary matrix representing the bands occurring in a set of DGGE/PFGE patterns. The presence or absence of bands in a sample is simply scored in a binary manner as 1 (present) or 0 (absent), relative to all of the bands detected in a set of DGGE/PFGE patterns. The binary data can then be presented in a dendrogram where the differences in fingerprint patters are represented in a graphical format or as a dendrogram using a distance-based cluster analysis techniques such as unweighted pairwise grouping with mathematical averages (UPGMA). Another possibility is to use multidimensional scaling (MDS) to reduce a complex fingerprint pattern to a point in a two-dimensional space (Van Hannen et al. 1999a). It is important to note that these types of analyses depend on consistency when detecting bands, and subjective determination of presence or absence should be avoided. Luckily, most commercially available gel analysis software programs allow researchers to use automated band detection parameters (e.g., band width and intensity), or even set their own thresholds for each parameter. As an alternative to comparisons based on presence or absence, the overall pattern of gel lanes can be compared directly using densitometry profiles (i.e., the pixel intensities at discrete positions in the gel). This type of analysis is based on pairwise correlations of profiles and can be used to avoid biases associated with band detection or loading unequal amounts of DNA. For densitometry profile analyses, each lane profile is compared to the others, and the resulting matrix of correlation values correspond to lane similarities. The correlation values can then be transformed to dissimilarity values (1 – similarity) that can be used for cluster analysis via UPGMA. <br /><br />Quantitative analysis of DGGE and PFGE gels is also possible. For DGGE, only semiquantitative analysis is possible, since individual band intensities cannot be used to infer target abundances in natural samples because differences in band intensity can arise from variable amplification efficiencies for different targets, and/or differences in background DNA. Thus fingerprint patterns can be compared, but individual band intensities should not be used to infer target abundance in natural samples. For PFGE, quantitative analysis involves measuring the relative fluorescence of each band. Based on this information, it is possible to obtain values for richness and abundance that can be used to calculate diversity indices. This type of analysis can be conducted using the commercial software noted above. Of course, it should be noted that both DGGE and PFGE are subject to a number of confounding errors. These sources of error should be carefully considered when deciding what types of analyses or comparisons should be conducted and not the least when the outcome of the analysis is interpreted.<br /><br />

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