Exercise 5.

T-RFLP

5. Terminal-Restriction Fragment Length Polymorphism (T-RFLP)

BACKGROUND Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis is a method of comparative community analysis. T-RFLP analysis is based on the restriction endonuclease digestion of fluorescently end-labelled PCR products (in our case the 16S rRNA gene). The digested products are separated by gel electrophoresis and detected on an automated sequence analyser. The method provides distinct profiles (fingerprints) dependent on the species composition of the communities of the samples. We apply T-RFLP to analyse the bacterial community in the gastrointestinal tract of pigs in order to investigate if the community responds to different feeding strategies (i.e. liquid feed versus dry feed), changes in feed composition, different feed structure (i.e. fine versus coarse, pelleted versus non-pelleted) or to the administration of feed additives (i.e. organic acids). An example is shown in Fig 1.

Fig. 1. T-RFLP profiles of the bacterial community in the stomach of two pigs fed either a coarse (A) or fine (B) non-pelleted diet. Each peak represents one or several bacterial populations. This figure shows an example of how feed structure has an influence on the diversity of bacteria in the stomach of pigs.

Other questions can be addressed by the T-RFLP fingerprinting method, i.e. 1. Is there a special mucus-associated microbial community? 2. Are fecal samples representative of the intestinal microbiota? 3. Does each individual harbour its own, specific microflora? 4. Which known intestinal bacteria are represented by the T-RFs? 5. Can we detect a succession of bacteria around weaning?

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Exercise 5. T-RFLP

The T-RFLP method is outlined in Figure 2 and includes DNA extraction/purification followed by PCR amplification. The primer set is usually a fluorescently 5-labelled forward primer annealing to the 3 end of the antisense strand and an unlabelled reverse primer annealing to the 3 end of the sense strand of the template marker gene (here the 16S rRNA gene). The PCR reaction thus results in double stranded DNA fragments all labelled in the 5 end of the sense strand.

Fig. 2. Outline of the T-RFLP -method The PCR reaction is followed by restriction enzyme digestion, whereby each DNA fragment renders one labelled terminal fragment and one or more unlabelled fragments. The DNA fragments are then separated (1 to 2 bases, depending on the total fragment length) by electrophoresis on the ABI sequence analyser, where the labelled fragments are recognised by the fluorescence detector. Internal standards (labelled fragment length markers) are included in each sample. The resulting chromatogram reveals the size-fragments present in the sample as well as the relative quantitative distribution among them. It is thereby possible to compare samples according to the presence/absence of peaks as well as relative distribution among the peaks.

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Exercise 5. T-RFLP

EXPERIMENTAL FLOW Day 1 Step 1. Sampling (1 hour). Day 2 Step 2. Preparation (stomaching) of samples (1 hour). Step 3. DNA extraction using bead beating and Geneclean kit (3 hours). Step 4. PCR with two primers to amplify the complete 16S rRNA gene - 4 PCR reactions per sample (1 hour). Day 3 Step 5. Purification and pooling of the PCR products using QIAquick kit. Agarose gel electrophoresis (2 hours). Step 6. Restriction enzyme digestion of PCR-products (1 hour + overnight). Day 4 Step 7. Preparation of T-RFs for electrophoresis (1 hour). Step 8. Electrophoresis of the T-RFs on the ABI automatic sequence analyser (1 hour + overnight). Day 5 Step 8. Data handling (4 hours). Appendix. Reagents and equipment. You end up with the following storage samples. 1. From step 1: samples (2 g) of digesta (to be stored at -200C), from which all are used in step 2. 2. From step 3: 50-l samples of extracted genomic DNA (to be stored at -20C), from which 4 x 2 l samples are to be used for Step 4. 3. From step 5: 50-l samples of QIAquick-purified PCR-amplified 16S rDNA (to be stored at 20C), from which 10 l samples are to be used for Step 6 4. From step 6: 20-l samples of T-RFs (to be stored at -20C), from which 2 l samples are to be used for Step 7. During the process you will have the following intermediary samples. 1. From step 2: 0.5-ml samples of diluted digesta (can be stored at -20C). The whole sample has to be used for DNA extraction in Step 3. 2. From step 4: four replicate 50-l samples of PCR-amplified 16S rDNA (can be stored at 20C), from which all are to be used for purification in Step 5.

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Exercise 5. T-RFLP

Step 1. Sampling (1 hour) 1. Exactly 2 grams of digesta from the stomach (sto), last third of the small intestine (SI3), caecum (cae) and mid third of the colon (Co2) are collected in small plastic bags (stomacher bags). The samples are kept on ice until storage at -200. Step 2. Preparation of samples (1 hour) 1. Dilute the samples 10X by adding 18 ml of sterile PBS buffer (pH 7.2) to the plastic bags. 2. Stomach the diluted samples for 2 min. in the bag mixer (Interscience). 3. Transfer 0.5 ml of sample into a sterile Eppendorf tube (can be stored at -200 C until DNA extraction - step 3). 4. Also transfer 0.5 ml of PBS buffer into a sterile Eppendorf tube and treat it as the samples (control of background) Step 3. DNA extraction (3 hours). 1. Add 5 l lysozyme (10 mg per ml freshly prepared if possible) to each of the samples, shake gently (i.e. turn the tube up and down once or twice) and incubate at 37C (thermo block) for 30 min. 2. Add 5 l Proteinase K (10 mg per ml) and 5 l RNase (10 mg per ml). Vortex and incubate at 65C (thermo block) for 1 hour. The increased temperature inhibits non-specific nucleases that would otherwise degrade DNA. 3. Add 50 l 24% SDS, vortex and incubate further 10 min at 65C. When the cells lyse, the suspension will clear up. 4. While the lysis suspension cools down, add approx. 0.5 g glass beads (diam. 0.18 mm) to bead beat vials. 5. Pour the lysis suspension into the vial and bead-beat for 3 min. 6. Centrifuge (13.000 rpm, 10 min). 7. Prepare the Geneclean Turbo Purification columns by placing the filter unit in the collection tube. Write the sample name/number on the filter unit. 8. Transfer (use pipette) the supernatant (approx. 550 l) from the bead beat vial directly to the Geneclean column. 9. Close the filter lid and centrifuge the column (13.000 rpm, 1 min). 10. Separate filter and collection tube. Empty the collection tube (The DNA is bound to the filter matrix). 11. Sample filter and collection tube again and add 500 l wash-solution to the filter through the hole in the lid (do not open the lid). 12. Centrifuge the column (13.000 rpm, 1 min). 13. Separate filter and collection tube. Empty the collection tube. 14. Sample filter and collection tube again and centrifuge the empty column (13.000 rpm, 4 min) in order to dry the filter. 15. Remove the lid from the storage tube and place the dry filter in the storage tube. Write sample name/number on the storage tube. 16. Add 50 l eluation solution to the centre of the filter through the hole in the lid. 17. Leave the tube on the desk for 5 min. 18. Centrifuge (13.000 rpm, 1 min). 19. Remove the filter from the tube and close the storage tubes with the lid. 20. The extracted DNA is stored at -20C and should always be kept on ice when used on the bench.

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Exercise 5. T-RFLP

Step 4. PCR-amplification of 16S rDNA (1 hour + 2-3 hours run). Four replicate 50-l PCR reactions are made for each sample. After amplification, the four samples are combined into one sample of 50 l. This provides a high concentration of PCR product which after digestion with the restriction enzyme (step 5) result in sufficient T-RFs for detection. In addition, the combination of multiple PCRs may reduce the significance of possible tube-to-tube variation of individual amplification reactions. PCR reaction master mix Nucleotides dNTP Primers (see appendix Exercise 6): FAM-S-D-Bact-0008-a-S-20 PHr DNA Polymerase: DyNAzyme II (Finnzymes) PCR buffer from kit H2O Mastermix DNA from Step 3 Total volume NB! Everything should be kept on ice until the PCR reaction is started. The master mix (minus DNA) is made up in a volume sufficient for all reactions and is dispersed with 48 l in each tube/well. The 2 l DNA can then be added. Remember to include in the mastermix a volume to a negative control for the PCR reaction (mastermix with no DNA added). The PCR reaction can run in single tubes or in 96-well PCR plates. The reaction is performed on a Hybaid PCR Express thermo-cycler using the following program (named "T-RFLP"). Initial incubation Melting of DNA strands Annealing of primers Primer extension Fimnal extension Termination 95C: 60 sec 95C: 30 sec 57C: 30 sec 72C: 45 sec 72C: 10 min 4C: (ca. 2-3 hours) 30 cycles (Ideally 10 ng per ml) (5 nmol per l) (5 pmol per l) (5 pmol per l) (2 U per l) (10) 2.0 l 2.0 l 2.0 l 0.5 l 5.0 l 36.5 l 48.0 l 2.0 l 50.0 l

The PCR-amplified samples of 16S rDNA can be stored at -20C, until purification is performed.

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Exercise 5. T-RFLP

Step 5. Purification of the PCR product with Qiagen purification kit (QIAquick) and agarose gel electrophoresis (2 hours) Purification. The four replicate fluorescently labeled PCR products from Step 4 are purified and combined using a QIAquick PCR Purification Kit (QIAquick Single Spin Coloumns, cat. No. 28106) QIAquick PCR purification kit protocol. 1. Add 250 l (5 volumes) Buffer PB to each of the four replicate 50 l (1 volume) PCR samples from step 4 and mix. 2. Place a QIAquick spin column in the 2 ml collection tube. Write sample name/number on top. 3. To bind the DNA, apply the four replicate PCR samples to one column and centrifuge (13000 rpm, 1 min). DNA is now bound to the column. 4. Discard flow-through solution from collection tube. Place the QIAquick column back into the same tube. 5. To wash the DNA, add 750 l Buffer PE to the QIAquick column and centrifuge (13000 rpm, 1 min). 6. Discard flow-through solution again and place the QIAquick column back into the same tube. 7. Centrifuge (13000 rpm, 1 min) the empty tube to remove residual ethanol/buffer from the column. 8. Place the QIAquick column in a clean 1.5 ml Eppendorf tube. Write sample name/number on tube. 9. To elute DNA, add 50 l Buffer EB (10 mM Tris-Cl, pH 8.5) to the centre of the QIAquick membrane and centrifuge (13000 rpm, 1 min). 10. The approx. 50 l QIAquick-purified 16S rDNA samples are stored at -20C. Samples of 10 l is used for the restriction digest (step 6). The purified and combined PCR amplified 16S rDNA are verified by electrophoresis on 1% agarose gel. Agarose gel elctrophoresis 1. Mix 100 ml TBE-buffer with 1 g agarose and boil the suspension in the microwave oven (max power, 2 min). 2. Let the solution cool a bit on the desk and add 5 l ethidium bromide solution (1%). Pour the solution into the electrophoresis chamber and place the comb in the solution. 3. When the gel is solidified, TBE is poured into the chamber and the comb is removed from the gel. 4. Gently mix samples of 2 l DNA with 0.5 l loading buffer on a piece of Parafilm. 5. Load 2 l of the samples into the lanes on the agarose gel. 6. Run the electrophoresis 4-5 min at 200 V. The blue indicator band should run approx. 0.5 cm into the gel. 7. Check the DNA bands on the UV table. Step 6. Restriction digestion of PCR-products (1 hour + overnight). Fluorescently labeled terminal restriction fragments (T-RFs) are produced by digestion using the restriction endonuclease HhaI (R0139L, BioLabs) with the recognition site 5'..G CGC..3'. 3'..CGC G..5'
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Exercise 5. T-RFLP

Restiction digest master mix Restriction endonuclease HhaI NE Buffer H2O PCR-product/ 16S Rdna

(20U/ ml) 10 Master mix volume

1 l 2 l 7 l 10 l 10 l

Total volumen

20 l

The master mix (minus PCR-product) is made up in a volume sufficient for all reactions and is dispersed with 10 l in each tube/well. The 10 l 16S rDNA (PCR product) is then added. The restriction digestion is performed in PCR single tubes or in 96-well PCR plates. The samples are incubated overnight at 370 C (can be performed in the Hybaid PCR Express thermo-cycler - use the program named "37C"). The T-RFs can be stored at -20C until electrophoresis on the ABI sequence analyser Step 7. Preparation of T-RFs for electrophoresis (1 hour). The T-RFs are mixed with an loading buffer and a standard size marker (GS-1000 ROX, PE Biosystems). Standard size marker Loading buffer T-RFs Total volumen 0.5 L 2.5 L 2 L 5 L

Again, a master mix (minus T-RFs) is made up in a volume sufficient for all samples and is dispersed with 3 l in each well of a 96-well PCR plate and then the 2 l T-RF is added. For denaturation of the DNA, heat the tubes (94C, 2-4 min) in the PCR machine. Transfer the tubes immediately thereafter to ice and keep them there until the samples (approx. 2 l) are loaded on the gel (see step 8). Step 8. Electrophoresis of the T-RFs on the ABI automatic sequence analyser (1 hour + overnight). The following is a description of the procedure for preparing the acrylamide gel and setting up the ABI automatic sequence analyser. The technician will do this step, but for your information we include it here anyway. 1. Wash glass plates (front plate (with cut and ground joint) and back plate). Be especially careful to clean the side with the ground joint (front plate) and the side with reversed writing (back plate), since these sides are facing the gel directly. Therefore use a) soap + hot water; b) deionised water from tap; c) Elga water. 2. Sampling of the plates. Place the back plate (no ground joint) in the bottom of the frame with the reversed writing upwards. Moisten the spacers (white plastic) and place them to follow the edge of the glass plates (the cut in the spacers should turn inwards and upwards). Place to front plate (ground joint downwards) on top of the other and tighten the clamps along the sides.

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Exercise 5. T-RFLP

3.

4.

5.

6.

7.

Mount the gel-pulling aggregate. Check that it is neither wet nor dirty and that the hole for the syringe is clear. Mount the syringe (dry and clean) and place the plates in the fume hood. Prepare an acrylic amide gel matrix by following the instructions on the package. The content is poured into a beaker glass and then directly into the syringe. Knock on the glass plate as the front of the gel advances to avoid air bobbles to get stocked in the gel. Mount the comb with the backside (no teeth) towards the gel and place clamps on the glass plate around it. The gel should now solidify for at least 1 hours. The gel plates are now cleaned to remove acrylic amide. This will otherwise burn into the plates. Be careful to clean around the black cross pin, where the laser light from the reader will penetrate. The comb is removed and the well is rinsed with Elga water. The comb is cleaned in water and placed in the well with the teeth pressed approx. mm into the gel. Place the bottom buffer chamber and the gel in the ABI. If the folder RA.MAIN4 is opened, close it and log the network connection off. In ABI Prism choose New Genescan Run, and run a plate-check (peaks = dirt on the glass plates where the laser light should penetrate). If any peaks, clean again the plates around the black cross pin before running another plate check. In the meanwhile, prepare 1400 ml TBE buffer. When the plate-check is OK, place the upper buffer chamber in the ABI and connect the wires. Fill up the upper chamber with buffer (to the white edge of the comb) Check that the chamber is tight. Then fill up the lower chamber. Mount the heating plate and connect power and water. Close the door. Pre-run. ABI Prism is started (Collection Genescan Run new.). Settings are checked (according to Anettes book). A prerun is started, where the temperature will increase to 51C.

While the temperature stabilises, the sample sheet is opened (new Sequence Sample). The sample sheet is named and saved. Then prepare the samples. Loading of the gel 1. Check that the temperature is at least 48C and pause the ABI (press OK!). The gel is loaded with 1.5-l samples in each lane using a multipipette. The samples are placed on the gel according to the sample sheet prepared in advance. Every second (uneven) lane is loaded first. The machine is closed and started (press Resume), and runs for approx. 2 min. Pause again and load the even lanes. Then cancel the pre-run, install your sample sheet and start the run. Step 9. Data handling (4 hours). 1. The result of the ABI run (Run Folder) is saved in the Ole or the Lene folder under Sequences being analysed on the BioMol network. 2. The Run Folder is copied to the desktop. 3. The Run folder is opened. The gel picture is opened in Gene Scan. The gel picture is checked visually and the tracking is evaluated. If the 96 lanes are not tracked correctly you have to perform a manual tracking - if possible - followed by a new extraction of the data. 4. When extraction is performed, switch of the Autoanalyse data. In Analysis Control, choose 'untitled' under Parameters and 'Test 5' under Standard and then click on Analyse. REFERENCES Clement, B.G., L.E. Kehl, K.L. DeBord and C.L. Kitts. 1998. Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J. Microbiol. Methods 31:135:142.

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Exercise 5. T-RFLP

Dunbar, J., L.O. Ticknor and C.R. Kuske. 2000. Assessment of microbial diversity in four southwestern united states soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66:2943-2950. Dunbar, J., L.O. Ticknor and C.R. Kuske. 2001. Phylogenetic specificity and reproducibility and new method for analysis of terminal restricttion fragment profiles of 16S rRNA genes from bacterial communities. Appl. Environ. Microbiol. 67:190-197. Kaplan,C.W., J.C. Astaire, M.E. Sanders, B.S. Reddy and C.L. Kitts. 2001.16S ribosomal DNA terminal restriction fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol. 67:1935-1939. Leser, T.D., R.H. Lindecrona, T.K. Jensen, B.B. Jensen and K. Mller. 2000. Changes in bacterial community structure in the colon of pigs fed different experimental diets and after infection with Brachyspira hyodysenteriae. Appl. Environ. Microbiol. 66:3290-3296. Marchesi, J.R., T. Sato, A.J. Weightman, T.A. Martin, J.C. Fry, S.J. Hiom et al. 1998. Design and evaluation of useful bacterium-specific PCR primers that amplify genes coding for bacterial 16S rRNA. Appl. Environ. Microbiol. 64:795-799. Marsh, T.L. 1999. Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diverstiy among homologous populations of amplification products. Cur. Op. Microbiol. 2:323-327. Moeseneder, M.M., J.M. Arrieta, G. Muyzer, C. Winter and G.J. 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. Osborne, 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 structure and dynamics. Environ. Microbiol. 2:39-50. Tiedje, J.M., S. Asuming-Brempong, K. Nsslein, T.L. Marsh and S.J. Flynn. 1999. Opening the black box of soil microbial diversity. Appl. Soil Ecol. 13:109-122. REAGENTS AND EQUIPMENT Step 1. Plastic bags labelled with sample number ect. Balance for weighing Step 2. . PBS buffer (8.5 g NaCl, 0.3 g KH2PO4, 0.7 g Na2HPO4 2H2O, pH 7.2) Bag mixer 1000-l pipette and tips Sterile Eppendorf tubes Step 3. Pipettes and tips Thermoblock 37C, 60C
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Exercise 5. T-RFLP

Lysozym, 10 mg per ml (-20C) Proteinase K, 10 mg per ml (-20C) Rnase, 10 mg per ml (-20C) SDS, 24% (RT) Glass beads (diam. 0.18 mm) and bead-beat vials Bead-beater apparatus Geneclean kit Eppendorf centrifuge Sterile Eppendorf tubes Step 4. Box with ice Pipettes and filter-tips Sterile Eppendorf tube (for preparation of the master mix) PCR vials and caps dNTP solution (5 nmol/l) Primers (5 pmol/l) Polymerase (2U/l) PCR buffer (10X) Sterile-filtered Elga water PCR-machine Step 5. Pipettes and tips QIAquick Turbo Purification Kit (columns, tubes, buffer) Eppendorf centrifuge Eppendorf tubes TBE-buffer (10.8 g Tris-base, 5.5 g boric acid, 40 ml 0.5M EDTA (pH 8.0) - usually a 10 concentrated form is made, which is diluted 1:9 in Elga water before use) Agarose Microwave oven Ethidiumbromide solution (1%) Gel electrophoresis system Loading buffer for agarose gel Parafilm UV-table Step 6. Eppendorf tube PCR tubes and caps Restriction endonuclease HhaI (20U/ml) NE Buffer (10X) Elga water Incubator, 370C Step 7. 96-well PCR plate GS-1000 ROX (standard size marker) Loading buffer PCR-machine (for denaturation)
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