Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis - PubMed (original) (raw)

Comparative Study

Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis

M M Moeseneder et al. Appl Environ Microbiol. 1999 Aug.

Abstract

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.

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Figures

FIG. 1

FIG. 1

(a) Migration behavior of the Texas red standard mixture when run at 3 kV for 75 min: 1, 75 bp; 2, 100 bp; 3, 154 bp; 4, 200 bp; 5, 220 bp; 6, 221 bp; 7, 296 bp; 8, 300 bp; 9, 344 bp; 10, 396 bp; 11, 400 bp; 12, 500 and 504 bp; 13, 517 bp; 14, 600 bp; 15, 700 bp; 16, 800 bp; 17, 900 bp; 18, 1,000 bp; 19, 1,632 bp. (b) A second-order polynomial regression was applied to determine the unknown fragments with these standards (n = 20). Unlabeled small peaks are unspecific peaks due to slight impurities or partial degradation.

FIG. 2

FIG. 2

(a) Screening for 16S rDNA PCR products from the South Aegean Sea (msb1). The calculated length of the 16S rDNA product is 1,503 ± 8 bp (n = 32). (b) T-RFLP fingerprinting of a complex marine bacterial community from the South Aegean Sea (msb1) cut with _Cfo_I for 6 h. Thirty-two of 44 T-RFLP OTUs were found between 20 and 517 bp. The largest fragments were ≈1,000 bp.

FIG. 3

FIG. 3

DGGE pattern from the samples taken in the Aegean Sea. (a) Banding pattern of the original gel stained with GelStar (FMC Bioproducts), acquired with a charge-coupled device camera over a 2-min exposure period. Longer integration times enhanced the signal from weak bands, thereby oversaturating stronger bands. (b) The DGGE gel was acquired with three different integration times, and the bands were drawn in a schematic diagram. Lanes: 1, msb3 (35 OTUs); 2, msb1 (35 OTUs); 3, mnb3 (36 OTUs); 4, mnb1 (33 OTUs); 5, mnb2 (33 OTUs); 6, mnb4 (28 OTUs).

FIG. 4

FIG. 4

UPGMA dendrograms from T-RFLP and DGGE analyses of PCR products of samples taken in the Aegean Sea. For the samples analyzed with T-RFLP, the numbers of OTUs from three separate restriction digests were pooled.

FIG. 5

FIG. 5

UPGMA dendrogram based on comparison of patterns from the upper mixed water column at different sampling sites. Designations: an, samples taken in the Middle Adriatic Sea off Ancona; msb, samples from the South Aegean Sea; mnb, samples from the North Aegean Sea; ro, samples taken in the Northern Adriatic Sea off Rovinj. Samples an4-1 and an4-2, as well as an60-1 and an60-2, are replicates from the same template. For samples msb1rt, mnb1rt, and mnb2rt, rRNA was reverse transcribed and used as the template in the PCR.

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