Article Figures & Data
Figures
- FIG. 1.
Impact of fluorophore incorporation on DGGE fingerprints. This SYBR Green I-stained gel contains PCR products from three soils with and without fluorophore labels. An asterisk indicates the position of two gradient-sensitive bands in the S14 fingerprints that appear either singly or as two closely associated bands. This fingerprint variation is not fluorophore specific and simply reflects the challenge of obtaining reproducible fingerprints using DGGE. Instead of 100 ng as for the other samples, only 50 ng of FAM-labeled samples was run in this gel to equalize signal intensities since FAM fluoresces at the same excitation wavelength as SYBR Green I. The amount loaded did not affect fingerprint migration (data not shown).
- FIG. 2.
Comparison of sample signal-to-gel background ratios for fluorophore-labeled and unlabeled PCR product dilutions. Error bars represent 95% confidence intervals for the ratios and were calculated from the standard deviation of the background signals (noise). Unlabeled products were stained with SYBR Green I and detected with either a Typhoon imager (SYBR) or a UV transilluminator (SYBR UV). Inset: Example of a PCR product dilution series (Cy5) indicating the band selected for quantification. This band represents approximately 2.5% of the total fingerprint intensity. While image intensity has been increased for this figure, the original image contains clear patterns for all samples without any saturated pixels.
- FIG. 3.
Comparison of interlane and intralane normalization using fluorophore-labeled standards. (A) Images of duplicate gels scanned for either Cy5 (samples) or FAM (standards). Triplicate samples were arranged in an alternating S4, S10, and S14 order in two gels. (B) UPGMA dendrograms of Pearson correlation matrices generated by interlane normalization. (C) UPGMA dendrograms of Pearson correlation generated by intralane normalization. Fingerprint ordering is identical between the two dendrograms from top to bottom to facilitate comparison.
