Extraction and Purification of Nucleic Acids

Total DNA was extracted adopting the direct lysis method by using the FastPrep® System, a bead beater that allows to desorb microbial cells from soil particles and provides a large lysis yield. In combination with the commercial FastSPIN® kit



Fig. 8.1 Flow sheet of employed molecular techniques

for Soil (MoBio) this method gives a highly purified DNA from small amount (0.5 g) of soil.

One milliliter of sodium phosphate buffer and 0.1 ml lysis buffer were added to 0.5 g of soil in a lysing matrix tube. The soil slurry was homogenized for 30 s at 5.5 m/s, centrifuged at 14,000 x g for 30 s, and the supernatant transferred to a 2.0 ml microcentrifuge tube. 0.25 ml of protein precipitation solution was added and the tube shaken ten times by hand before centrifuging at 14,000 x g for 5 min to precipitate a protein pellet. The supernatant was transferred to a clean microcentrifuge tube in which 1 ml of binding matrix was added to bind DNA. After the setting of silica matrix for 3 min, about half of the supernatant was carefully removed, while the remaining part of the supernatant was resuspended in the binding matrix and then transferred to a SPIN filter to be centrifuged at 14,000 x g for 1 min. SPIN filter was washed twice with 0.5 ml SEWS-M, air dried for 5 min and the DNA eluted with 0.1 ml DNase free water.

A direct lysis method was also adopted to extract RNA, by using the commercial RNA PowerSoil™ Total RNA Isolation (MoBIO). Two grams of soil were added to a 15 ml Bead Tube. 3.5 ml of a mix of bead/lysis solutions were added to the tube and this was vortexed for 5 min before adding 3.5 ml of phenol:chloroform:isoamyl alcohol mixture (25:24:1, pH 7.0) and shaken again by vortex for 10 min. Water phase was obtained after centrifuging at 2,500 x g for 10 min and DNA ethanol precipitated, purified by a RNA Capture Column, eluted with 0.1 ml RNase free water and stored at —80°C before use.

8.2.3 Characterization of Extracted Nucleic Acids

DNA and RNA concentration was determined spectrophotometrically at 260 nm by the UV/visible NanoDrop® ND-1000, a spectrophotometer that used very low amounts of nucleic acid solution (1ml) and concomitantly enabled to determine the quality indices A260/A230 and A260/A280. Electrophoresis of nucleic acids on ethidium bromide stained 0.7% agarose gel was used to evaluate the average molecular weight (>10 kb), the presence of degradation smear for DNA, and the presence of the two major rRNA bands (0.9 and 1.5 kb for 16S and 23S rRNA, respectively).

8.2.4 DNase Digestion and Retrotranscription of RNA

Extracted RNA was digested with DNase to eliminate DNA traces eventually coextracted. Because single strand RNA does not constitute a valid template for PCR, RNA was retro transcribed to obtain the cDNA-RNA hybrid by using 100 U of the MMLV-RT enzyme and the RETROscript® kit (Ambion®), according to manufacturer' instructions.

8.2.5 PCR Amplification

Both DNA and cDNA-RNA hybrid extracted and purified from soil samples were amplified by PCR of conserved regions of the gene coding for the small subunit of ribosomal RNA. Eubacterial communities were investigated by using the 968F-1401R primers (Heuer and Smalla 1997), targeting the V6-V8 region of the 16S rDNA that, in Escherichia coli, spans between the 968 and 1,401 positions of the gene, and amplifying a fragment of about 450 bp (Brosius et al. 1978). In particular, the forward 968F primer was modified to contain a GC-clamp on the 5' end, a 40 G and C stretch that increases the melting temperature of the amplicons, thus avoiding their complete denaturation under denaturing gel electrophoresis (Muyzer et al. 1993). Reaction mix contained 20 ng DNA template, 3 U of Taq polymerase (Euroclone), 50 pmol of each of the two primers, 10 nmol each of dNTPs, 2.5 mM MgCl2, in a buffered final volume of 50 ml. Amplification conditions were as follows:

10 min at 72° C (final elongation step)

PCR fragments were checked by electrophoresis on ethidium bromide (0.5 mg/ml) stained 1.5% agarose gel.

8.2.6 Denaturing Gradient Gel Electrophoresis

In order to load the same amounts of amplicons onto gels free of unincorporated dNTPs and primers, PCR products were previously purified by the UltraClean™ PCR Clean-up Kit and quantitatively assayed by the NanoDrop ND-1000 spectrophotometer.

DGGE was performed by using the DCode™ Universal Mutation Detection System (Bio-Rad Laboratories, Hercules, CA, USA). 400 ng of PCR amplicons were loaded onto 6% (w/v) acrilamide (acrylamide, N,N'-methylenebisacrylamide, p/p, 37.5:1) vertical gel, with a "top-filling" linear gradient (that is, increasing denaturing conditions from the top to the bottom of the gel) ranging from 45 to 60% (100% corresponds to 7M urea - 40% v/v deionized formamide). Electrophoresis was performed for 15 h at 75 V 60° C in 1X TAE buffer (40 mM Tris base, 20 mM acetic acid, 1 mM EDTA, pH 8.3). Gels were stained in SYBR green I (Fluka), a nucleic acid gel stain that, once irradiated by UV lamps, emits fluorescent light at 520 nm. In order to compare gels run separately, even in the presence of a

large number of samples, a marker consisting of amplicons relative to the 16S rDNA of Lactobacillus sanfranciscensis and to the p subunit of RNA polymerase (De Angelis et al. 2007) were loaded.

Electrophoretic profiles were acquired by the Gel Doc System (Biorad) and digitally elaborated by the BioNumerics 4.5 software (Applied Maths). Fingerprints comparison and phylogenetic trees were by the UPGMA (unweighted pair-group method using arithmetic averages) method and the Pearson correlation coefficient (Rademaker and de Bruijn 2004) that, taking into account densitometry curves, is not affected by the typical drawbacks occurring when coefficients are determined by aligning gel bands (Boon et al. 2002; Krave et al. 2002).

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