Table 1. Primers and PCR conditions used in quantitative PCR† and DGGE# with GC clamp
ggcggcgcgccgcccgccccgcccccgtcgccc added to the 5‘ end*.
gene
16S
primer
sequence
331F#†
*
534r#†
att acc gcg gct gct gg
nirK876†
aty ggc ggv cay ggc ga
nirK1040
nosZ
tcc tac ggg agg cag cag t
qPCR primer
(nM)
reference
500
1
500
2
194
nirK
nirS
product
(bp)
†
5000
165
gcc tcg atc agr ttr tgg tt
nirKFIaCu#
atc atg gts ctg ccg cg
nirKR3Cu#
*gcc tcg atc agr ttg tgg tt
nirS_cd3a-F#†
gts aac gts aag gar acs gg
nirS_R3cd-R#†
3
5000
472
4
2500
5
*gas ttc ggr tgs gtc ttg a
2500
6
nosZ2R†
cak rtg cak sgc rtg gca gaa
5000
nosZ2F†
cgc rac ggc aas aag gts mss gt
nosZ1622RC#
*cgs acc tts ttg ccs tyg cg
#
nosZF
410
268
7
2500
6
453
cgy tgt tcm tcg aca gcc ag
To reduce heterogeneity between individual DNA extracts, each DNA sample was made from
two separate aliquots of 250 mg soil from each sieved core sample used, and then pooled.
For DGGE profiling duplicate PCR reactions were carried out for each of the three replicate
samples. PCR amplifications for DGGE were performed in 20µl volumes with 200 µM
dNTPs, 2.5 mM MgSO4, 0.2 µM Primers for 16S rRNA, nirK and nosZ and 0.4 µM for nirS
primers, 3% DMSO for nosZ and 0.5µg BSA for nirS and nirK. The 16S rRNA, nirK, nirS
and nosZ products were run at 60 °C on 8% acrylamide gels with 30-60%, 30-60%, 30-45%
and 30-50% denaturing conditions, respectively. DGGE PCR cycling conditions - initial
denaturing step 98oC (45sec) followed by 30 cycles: annealing temperature 64oC (30 sec);
8
extension temperature 72oC (30 sec); denaturing 98 oC (45sec). A final extension step at 72oC
(10 min) was included.
For qPCR analysis triplicate PCR reactions were carried out for each of the three replicate
samples. qPCR cycling conditions - initial activation for HotStarTaq DNA polymerase at
95oC (15 min) followed by 40 cycles: annealing temperature 58oC (18 sec); extension
temperature 72oC (42 sec); denaturing 95 oC (15sec). A final dissociation curve was
generated to determine the quality of the applified products.
References
1.
Nadkarni, M.A., Martin, F.E., Jacques, N.A. & Hunter, N. 2002. Determination of
bacterial load by real-time PCR using a broad range (universal) probe and primer set.
Microbiology 148, 257–266.
2.
Muyzer, G., Dewaal, E.C. & Uitterlinden, A.G. 1993. Profiling of complex microbial
populations by denaturing gradient gel electrophoresis analysis of polymerase chain reactionamplified genes coding for 16S rRNA. Appl Environ Microbiol 59, 695-700.
3.
Henry, S., Baudoin, E., López-Gutiérrez, J.C., Martin-Laurent, F., Brauman, A. &
Philippot, L. 2004 Quantification of denitrifying bacteria in soils by nirK gene targeted realtime PCR. J. Microbiol Methods 59, 327-335. (Erratum, 61: 289-290.) (DOI:
10.1016/j.mimet.2004.07.002; DOI: 10.1016/j.mimet.2004.12.008)
4.
Hallin S. and Lindgren, P.-E. (1999) PCR detection of genes encoding nitrite
reductase in denitrifing bacteria. Appl. Environ. Microbiol. 65, 1652–1657.
5.
Michotey, V., Méjean, V. & Bonin, P. 2000 Comparison of methods for quantification
of cytochrome cd1-denitrifying bacteria in environmental marine samples. Appl Environ
Microbiol 66, 1564-1571.
6.
Throbäck, I.N., Enwall, K., Jarvis, A. & Hallin, S. 2004 Reassessing PCR primers
targeting nirS, nirK and nosZ genes for community surveys of denitrifying bacteria with
DGGE. FEMS Microbiol Ecol 49, 401-417. (DOI: 10.1016/j.femsec.2004.04.011)
7.
Henry, S., Bru, D., Stres, B., Hallet, S. and Philippot L. 2006 Quantitative detection
of the nosZ gene, encoding nitrous oxide reductase, and comparison of the abundances of
16S rRNA, narG, nirK, and nosZ genes in soils. Appl Environ Microbiol 72, 5181-5189.
(DOI: 10.1128/AEM.00231-06)
8.
Kloos, K., Mergel, A., Rosch, C. & Bothe, H. 2001 Denitrification within the genus
Azospirillum and other associative bacteria. Aust J Plant Physiol 28, 991-998.