Single Strand Conformation Polymorphism (SSCP)

Bacterial denitrifying communities of the differently fertilized soils and the organic fertilizers were compared by the cultivation-independent fingerprinting technique “Single Strand Conformation Polymorphism” (SSCP) based on PCR amplification. Schwieger and Tebbe (1998) described for the first time the method derived from medical research for investigation of complex bacterial communities. PCR products were subjected to SSCP (Dohrmann and

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Tebbe 2004) to generate genetic profiles, which corresponded to the diversity of the amplified nirS gene fragments.

Single-strand DNA molecules fold under non-denaturing conditions into secondary conformations that are primarily caused by the sequence of bases since complementary bases tend to form hydrogen bonds thus forming the secondary structure. As a result of PCR with primers binding to a gene of many different organisms in a community, products are generated that have a very similar size but different base sequences. These conformations encounter differential impedance within a polyacrylamide gel matrix and can therefore be separated by electrophresis.

2.10.5.1 PCR Assays for SSCP

PCR for SSCP was processed using the nirS primers cd3aF (GT(C/G) AAC GT(C/G) AAG GA(A/G) AC(C/G) GG (Michotey et al. 2000)) and R3cd (GA(C/G) TTC GG(A/G) TG(C/G) GTC TTG A (Throbäck et al. 2004)) resulting in PCR fragments of 425 bp length. A modification of the reverse primer, i.e. phosphorylation at the 5’ end of R3cd, was necessary for subsequent single-strand digestion. PCR conditions comprising mastermix composition, amount of primers, annealing temperature and time, number of cycles, and “touchdown“ program had to be optimized in depencence of the used primers. Finally, PCR was performed in a total volume of 25 µl containing 1 x PCR buffer, 0.16 mM of each desoxy- nucleotide, 3 mM MgCl2, 2 U recombinant Taq Polymerase, 0.5 µM each primer (MWG Biotech, Ebersberg), and 0.25 µl bovine serum albumin (20 mg ml-1_{, Fermentas, St. Leon-Rot)} to enhance amplification of environmental DNA. The amount of template DNA was adjusted to 25 ng per reaction (2.10.1). DNA amplification was conducted in 6 replicates in the thermal cycler MyCycler (Bio-Rad, München). After a denaturation step of 3.5 min at 94°C, a “touchdown” PCR was performed that consisted for amplification of nirS fragments of a denaturation step of 30 s at 94°C, a primer-annealing step of 30 s, and an extension step of 60 s at 72°C. The annealing temperature was decreased by 0.5°C each cycle, starting at 63°C until it reached after 13 cycles a “touchdown“ at 57°C. Additional 19 cycles were processed at an annealing temperature of 57°C. After 32 cycles, a final extension step of 15 min at 72°C was performed. The correct length of the amplification products was controlled by staining DNA with ethidium bromide after electrophoresis on 1.7% [wt/vol] agarose gels (Peqlab, Erlangen).

2.10.5.2 Single-Strand Removal

PCR products (2.10.5.1) were pooled, purified (2.10.4), and quantified (2.10.3). Double- stranded DNA (approximately 700 ng) were converted to single-stranded products by selective removal of the reverse phosphorylated strands with 2.5 U lambda-exonuclease (New England Biolabs, Frankfurt/Main) in 1 x exonuclease buffer (provided by the

manufacturer) at 37°C for 45 min. Digestion was stopped by starting the purification of the remaining single-strands using the MinElute PCR Purification Kit (Qiagen, Hilden) according to the manufacturers instruction. Single-stranded DNA was eluted with 10 µl EB buffer included in the kit.

Prior to loading on the polyacrylamid gel the single strands had to be denatured. Therefore, DNA was after spiking with 10 µl of SSCP loading buffer (10 mM NaOH, 0.25% [wt/vol] xylene cyanol, 0.25% [wt/vol] bromophenol blue, 95% [vol/vol] formamide) heated to 95°C for 2 min and immediately cooled on ice.

2.10.5.3 SSCP Gel Casting and Electrophoresis

Since the physicochemical environment during electrophoresis has an enormous effect on the electrophoretic mobility and the resolution of SSCP, composition of the polyacrylamide gel, buffer strength, temperature, and running time had to be optimized carefully depending on the DNA fragment length and the used primer. The electrophoretic separation was performed with the Protean II XL vertical electrophoresis cell (Bio-Rad, München) and the appropriate cooling module.

Two glass plates (20 x 20 x 0.4 cm and 22 x 20 x 0.4 cm) were cleaned three times with 70% [vol/vol] ethanol and polished with lint-free tissues before gel-casting procedure started. Spacers and comb were cleaned likewise. The smaller plate was impregnated with a few drops of repel-silane (SEA-Spray, CLP, England) by a lint-free tissue, and after 2-3 min it was polished with a fresh tissue, rinsed subsequently with 70% ethanol and polished again. In contrast, the larger plate was impregnated with 1 ml of bind-silane use solution (10 ml 96% [vol/vol] ethanol + 100 µl bind silane (Amersham Biosciences, Freiburg) + 100 µl 100% [vol/vol] acetic acid) in the same way as described for the other plate. A sandwich of both glass plates and the 0.35 mm spacers were prepared and clamped in the casting stand. The SSCP gel matrix consisted of 0.725 x MDE gel solution (Cambrex Bio Science, Belgium), 10% [wt/vol] formamide, 1 x TBE buffer, 4% [wt/vol] APS (ammonium persulfate), and 0.04% [wt/vol] TEMED (N, N, N’, N’ - tetramethylethylenediamine). When APS and TEMED had been added to the gel solution (careful mixing with a magnetic stir bar, no bubbles should emerge) the gel had to be poured quickly between the glass plates without air bubbles. The comb was ca. 1 cm inserted inversely, i.e. with the linear border into the gel matrix. Afterwards, the gel polymerized at least 2 h at room temperature. Coolant circulation within the cooling core was already turned on and the temperature was adjusted to 19.5°C to ensure this constant temperature of the cooling system right at the beginning of the electrophoretic separation. After polymerization the comb was reversed, the plate sandwich was placed in the electrophoresis cell, and 1 x TBE buffer was poured into the vessel. Before some drops of the SSCP loading dye were transferred to the sample wells, they were rinsed with buffer using a syringe and a needle, and a “pre-run” of about 15 min was startet. Thereafter, the denatured

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samples were loaded into the wells (up to 8 µl per well), and the gel was run for 17 h at constant 450 V and 19.5°C.

2.10.5.4 Silver Staining of DNA

To visualize the SSCP-profiles, DNA within the polyacrylamide gels was silver-stained (Bassam et al. 1991) in carefully cleaned trays of stainless steel. The gel fixed to the glass plate was removed from the electrophoresis chamber, was transferred into a tray filled with 300 ml 10% [vol/vol] acetic acid and was incubated for 30 min under mild shaking. In the meantime, the staining solution (500 ml pure water + 0.5 g AgNO3 + 0.75 ml 37% [wt/vol] formaldehyde) and the developer (500 ml pure water + 10.43 g Na2CO3 anhydrous) were prepared and stored in darkness and at 4°C, respectively. After the DNA fixation, the gel was washed three times for 5 min with pure water. For color impregnation, the gel was incubated for 30 min in the staining solution protected from light and afterwards rinsed for 10 s with pure water. The cooled developer solution was completed by adding 1 ml 37% [wt/vol] formaldehyde and 0.5 ml 0.2% [wt/vol] sodium thiosulfate, and the gel was then transferred to another tray filled with a small amount of the developer to rinse the gel for 20 s. In the residual developer, the gel was incubated until the patterns became clearly visible. To stop the staining process, the gel was transferred back to the first tray filled with 300 ml 10% [vol/vol] acetic acid for 4 min. After the color fixation, the gel was incubated in pure water for 30 min and subsequently dried at room temperature. Finally, the gel was scanned using the PowerLook 1120 (UMAX, Willich) scanner.