Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

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.

Archaeal community structures in rice soils from different geographical regions before and after initiation of methane production

“…Molecular community ¢ngerprinting by T-RFLP analysis is not only used for phylogenetic/taxonomic information but also for analysis of relative proportions of dominant phylotypes within the microbial community [28,33,41,42]. Although the rDNA-based T-RFLP analysis su¡ers from the lack of ¢ne resolution at the species level, and also from biases related to cell lysis, DNA extraction and puri¢cation, and PCR ampli¢cation, it has many advantages for comprehensive sampling and rigorous comparative community analysis which can include process and biogeographical investigations [43,44]. We have used the T-RFLP ¢ngerprinting technique for identi¢cation and quanti¢cation of the major archaeal phylotypes in di¡erent rice soils.…”

Methods for Measuring Denitrification: Diverse Approaches to a Difficult Problem

“…Various techniques are used to assess abundance such as direct DNA probing (Linne von Berg and Bothe 1992), quantitative PCR or real-time PCR (Gruntzig et al 2001, Henry et al 2004, which measure gene copies and relates that back to cell numbers, or T-RFLP (terminally labeled restriction-length fragment polymorphism, commonly used for estimates of diversity), which has been shown to be semiquantitative (Liu et al 1997). Community-level information can be obtained by analysis of PCR products by T-RFLP (Liu et al 1997) or denaturing gradient gel electrophoresis; DGGE (Moeseneder et al 1999). These techniques provide a profile of the gene population that reflects the diversity and relative abundance of each of the target sequences and can be used to detect changes in community dynamics related to geographic distance and biogeochemical properties.…”

Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availability

“…DNA concentrations were determined by measuring the absorbance at 260 nm and purity was checked by determining 260/280 nm absorbance ratio. 16S rDNA was amplified from extracted DNA using universal primers 27F (5¢-AGA GTT TGA TCC TGG CTC AG-3¢; Lane, 1991) and 1492R (5¢-TAC CTT GTT ACG ACT T-3¢; Lane, 1991;Moeseneder et al, 1999). 27F was fluorescently labelled at the 5¢ end with the phosphoramidite dye 6-FAM (MWGspa BIOTECH).…”