July 2002

V. Grüntzig, B. Stres, H. L. Ayala del Río, and J. M. Tiedje
Center for Microbial Ecology, Michigan State University
East Lansing, Michigan, 48824

I. Overview of T-RFLP method for community studies
Terminal restriction fragment length polymorphism (T-RFLP) allows the fingerprinting of a community by analyzing the polymorphism of a certain gene. It is a high-throughput, reproducible method that allows the semi-quantitative analysis of the diversity of a particular gene in a community. The figure below illustrates the procedure and the rationale of the method.

The DNA is harvested from the analyzed sample (1). The gene of interest is amplified using the polymerase chain reaction (PCR) with a fluorescently labeled primer (2). This yields a mixture of amplicons of the same or similar sizes with a fluorescent label at one end. After purification, the amplicon mixture is digested with a restriction enzyme, which generates fragments of different sizes (A-F) (3). These are separated through gel or capillary electrophoresis (4). A laser reader detects the labeled fragments and generates a profile based on fragment lengths (5).

Two methods have been widely used for separation of the fragments obtained after enzymatic restriction of labeled PCR products: gel electrophoresis in polyacrylamide gels and capillary electrophoresis. The former has until recently been more widespread and most of the available protocols for T-RFLP have used this method. However, capillary electrophoresis has been gaining popularity, but the standard procedures for the capillary equipment are not suitable for T-RFLP. Therefore we present a protocol for improved T-RFLP by capillary electrophoresis. The key new steps are identified below as [NEW].

II. Protocol
Steps involved in T-RFLP analysis:
1) Extraction of community DNA from the sample of interest (soil, sediment, reactor material, water sample, etc). The DNA extraction method of user's choice should yield DNA of high quality lacking inhibitory compounds that could interfere with the subsequent PCR reaction.
2) PCR amplification of gene of interest with fluorescently labeled primers. The T-RFLP method can be applied to any gene, therefore the primer selection will depend on the user's interest. However, 16S rDNA is frequently the gene of choice for microbial community analysis (see PCR protocol). Protocols for specific genes of interest are available elsewhere in literature. The amplification can be performed using a fluorescently labeled forward or reverse primer. Both primers can also be labeled with a different dye and used simultaneously in the same reaction. It might be necessary to pool several PCR reactions to obtain enough product for further steps (200-300 ng of DNA recommended per restriction digest). The amplification efficiency of labeled primers tends to be lower than that of unlabeled primers, frequently leading to lower yields.
3) Concentration and cleaning of PCR products. Excess primers, salts and possible nonspecific PCR products are removed by gel purification. The volume of the pooled PCR reactions can be reduced to half to a fifth of the original volume using a Speedvac or ethanol precipitation in order to facilitate the loading of the sample. Gel purification is then performed by separating the PCR products by electrophoresis on agarose gel, excising the band of proper size and recovering the PCR product with a gel purification kit (Qiagen, MOBIO, Promega) following manufacturer's recommendations. If nonspecific PCR products are not detected, regular PCR clean up kits can also be used.
4) Restriction digestion of the PCR products. Once purified, the PCR products are digested with a restriction enzyme (a four base pair cutter is most appropriate as the probability of having a restriction site within the amplicon is high). Various restriction enzymes can be used in single-enzyme reactions in order to determine which one yields the highest number and most even distribution of terminal restriction fragments. For each digestion, 100-150 ng of purified PCR product (assuming a 50% loss during purification) and 10-20 U of restriction enzyme should be used. The incubation period at the enzyme's optimal temperature can vary from 4-12 h to assure complete digestion. Restriction enzymes are inactivated by heating to 65-80ºC for 20-25 min.
5) [NEW] Desalting of restriction digest. In capillary electrophoresis the injection of DNA samples can be achieved by two methods. First, hydrodynamic injection requires pressure difference over the capillary. Alternatively, electrokinetic injection uses a combination of electrophoresis and electroendosmosis to inject the sample. Applied Biosystems PRISM 310 and 3100 Genetic Analyzers (PE Biosystems) use the latter. The presence of ions can interfere with the uptake of DNA using electrokinetic injection because of preferential injection of higher charge-to-mass molecules (e.g. Cl¯ ions). Therefore it is essential to desalt the inactivated restriction digest with Microcon columns (Amicon), Qiaquick Nucleotide Removal Kit (Qiagen) or conventional ethanol precipitation. In our case, the restriction products were diluted with water up to 500 μl, before concentration and desalinization on Microcon columns (see figure below).
6) [NEW] Capillary electrophoresis (CE); loading. A fifth to a third of the desalted restriction digest is generally loaded onto the capillary electrophoresis system using the default settings optimized for sequencing. This can lead to insufficient fluorescent signal, therefore yielding a low number and height of peaks. The injection time and injection voltage can be varied to regulate DNA uptake. The default sample injection time is generally 10 sec, however longer injection times increase the uptake of DNA from dilute sample solutions, therefore yielding a higher fluorescent signal with more peaks detected. Furthermore, injection voltage is directly proportional to the amount of DNA injected and can be modified from the default of 3 kV. Further concentration of the desalted product may also be necessary to stay within the appropriate CE loading volume.

III. Results
The improved protocol is demonstrated for the denitrifier community structure of a marine sediment (Figure 2).

Figure 2. T-RFLP profiles from marine sediment samples. Nitrite reductase genes (nirS) were amplified from purified DNA and digested with Hhal. The digested PCR products were separated without previous desalting (A), and after desalting using Microcon columns (B) (Amicon). IT, injection time, TP, total number of peaks detected by the instrument.

Desalting the restriction digest increased the DNA uptake leading to a higher fluorescent signal from the different fragments. This also increased the total number of peaks detected, as more peaks lie now above the fluorescence threshold of 50 units (Figure A). Increasing the injection time, further increased the uptake of DNA, leading to an even higher fluorescence signal and number of peaks detected. In the desalted sample analyzed, the elongation of the injection time from 10 to 60 sec, led to a 3.5 fold increase in number of peaks (Figure 1B). Desalting the restriction digest and applying an injection time from 30 to 60 sec produced a profile similar to that obtained when the same sample was analyzed by T-RFLP using polyacrylamide gel electrophoresis. Hence we recommend 60 sec injections of desalted samples.

References and suggested readings:

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Braker, G., H.L. Ayala-del-Río, A.H. Devol, A. Fesefeldt, and J.M. Tiedje. 2001. Community structure of denitrifiers, Bacteria, and Archaea along redox gradients in Pacific Northwest marine sediments by terminal restriction fragment length polymorphism analysis of amplified nitrite reductase (nirS) and 16S rRNA genes. Appl. Environ. Microbiol. 67(4): 1893-1901.

Brunk, C.F., E. Avaniss-Aghajani, and C.A. Brunk. 1996. A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Appl. Environ. Microbiol. 62: 872-879.

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Giovannoni, S. 1991. The polymerase chain reaction, p. 177-203. In E. Stackebrandt, and M. Goodfellow (ed.), Nucleic acid techniques in bacterial systematics. J. Wiley & Sons Ltd., West Sussex, United Kingdom.

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Moesender, M., J.M. Arrieta, G. Muyzer, C. Winter, and G. Herndl. 1999. Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 65: 3518-3525.

Osborn, A.M., E.R.B. Moore, and K.N. Timmis. 2000. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2:39-50.

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