DESCRIPTION OF DGGE AND FUNCTIONAL GENE DETECTION:
DGGE analysis: For day 281 and 310 samples the following PCR conditions were followed.
Each 30 µl reaction volume had the following final concentrations: 0.5 ng/µl DNA, 0.5 µM of
each primer: 341F (5’-CC TAC GGG AGG CAG CAG-3’ containing a 40-bp GC clamp at the 5’
end) and 786R (5’-CTA CCA GGG TAT CTA ATC-3’) (Baker et al. 2003), 2 mM MgCl2, 1x
PCR buffer, 0.25 mM of each dNTP and 0.08 U/µl Taq DNA polymerase, 400 ng/µl BSA. The
following PCR program was used: 95ºC for 3 min; 30 cycles of 94ºC for 30 s, 56ºC for 30 s,
72ºC for 30 s; one cycle of 72ºC for 5 min on a MJ Research Peltier Thermal Cycler PTC-200.
Triplicate PCR products were pooled to reduce amplification bias and cleaned using a
QIAquick® PCR purification kit (Qiagen Inc., Valencia, CA). The expected fragment was
visualized on 1% agarose gels by staining with ethidium bromide. Total community fingerprint
analysis was performed with the DCode Universal Mutation Detection System (BIO-RAD,
Hercules, CA). DGGE gels were created with a 30 to 50% denaturing gradient. The PCR product
was mixed with 2X loading dye and 20 µl loaded onto the gel. The samples were run for 16
hours at 80V in 1X TAE buffer preheated to 60ºC. The gels were stained for one hour with
ethidium bromide and visualized using Epi Chemi II darkroom (UVP, LLC, Upland, CA).
Dendrograms were created with Gelcompar II (Applied Maths, Inc., Austin, TX) using the
Pearson correlation coefficient and the unweighted pair group method with arithmetic mean
(UPGMA) clustering algorithm. Bands of interest were excised from the gel and DNA was
eluted in PCR-grade water overnight. DNA was PCR amplified in triplicate with the same
amplification reaction as described above, with the exception that the forward primer did not
have the GC clamp. Amplification products were pooled, cleaned, amplified with
BigDye®Terminator V3.1 cycle sequencing kit (Applied Biosystems Inc., Foster City, CA) and
submitted for sequencing. Partial nucleotide sequences were submitted to GenBank.
Functional Gene Detection: Samples of pore water and matrix from day 281 and day 310 were
tested for the presence of the functional genes, pcrA and cld, using PCR amplification. The pcrA
and cld genes were amplified from total DNA in triplicate PCR reactions. The cld gene was
amplified as outlined by Bender et al. (2004) with the exception that the PCR reactions were
carried out in 30 µl reactions. The pcrA gene was amplified in a 30 µl reaction volume with the
following final concentrations: 0.5 ng/µl DNA, 0.4 µM of each primer: pcrAF 5’ACTACATGTATGGNCCGCATCG-3’ and pcrAR 5’-CGTGRTCRCYGTACCAGTCRAA-3’,
1.5 mM MgCl2, 1x PCR buffer, 0.20 mM of each dNTP and 0.05 U/µl Taq DNA polymerase,
250 ng/µl BSA. The following PCR program was used: 94ºC for 2 min; 30 cycles of 94ºC for 30
s, 55ºC for 30 s, 72ºC for 1 min; one cycle of 72ºC for 10 min on a MJ Research Peltier Thermal
Cycler PTC-200. Triplicate PCR products were pooled to reduce amplification bias and cleaned
using a QIAquick® PCR purification kit Qiagen Inc., Valencia, CA). The two functional genes
were sequenced and closest relatives identified as previously outlined. Multiple sequence
alignments were created using the program ClustalX, version 1.83 (Thompson et al., 1997) and
phylogenetic analyses were conducted using the software package MEGA version 4 minimum
evolution analysis, using the Tamura–Nei model, with bootstrap values of 1,000 replicates
(Tamura et al., 2007). Partial nucleotide sequences were submitted to GenBank.
References:
Baker, G.C., Smith, J.J., and Cowan, D.A. (2003) Review and re-analysis of domain-specific 16S
primers. J Microbiol Methods 55: 541– 555.
Tamura K, Dudley J, Nei M, Kumar S. (2007) MEGA4: Molecular evolutionary genetics
analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. (1997) The Clustal X
windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res 24:4876–4882.