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2
Supplementary material
Table S1:
3
Applied primer systems, DGGE gradient and acrylamide concentration (AA) (Ref. = Reference)
Primer
Target Gene
[Function]
Sequence
341f*
907r
803
16S rDNA
Bacteria
Me1f#
Me2r
Me3f*
(500 bp)
Methyl-coenzyme
GCM ATG CAR ATH GGW ATG TC
A reductase (mcrA) TCA TBG CRT AGT TDG GRT AGT
[Methanogenesis]
ATG TCN GGT GGH GTM GGS
TTY AC
CCT ACG GGA GGC AGC AG
CCG TCA ATT CMT TTG AGT TT
CTA CCA GGG TAT CTA ATC C
Cycler Program
Ref
95°C 5 min; 1 min 95°C, 1
[1–3]
min 55°C, 1 min 72°C (35x);
10 min 72°C
95°C 5 min; 45 s 95°C, 45 s
50°C, 1.5 min 72°C (35
cycles); 10 min 72°C
Gradi
ent
AA
40-65
7%
[5, 6]
40-70
8%
# 95°C 5 min; 45s 95°C,
45s 54°C, 45s 72°C (40x);
10 min 72°C
nirS cd3af
nirS3cdr**
(500 bp)
Cytochrome cd1depending nitrite
reductase (nirS)
[Denitrification]
GTS AAC GTS AAG GAR ACS GG
GAS TTC GGR TGS GTC TTG A
95°C 10 min; 45 s 95°C,
45s 60°C, 45 s 72°C (40x);
10 min 72°C
[7]
40-70
8%
nirK laCuf Copper-depending
nirK3Cur** nitrite reductase
(nirK)
(450 bp)
[Denitrification]
ATC ATG GTS CTG CCG CG
GCC TCG ATC AGR TTG TGG TT
95°C 10 min; 45 s 95°C,
45s 65°C, 45 s 72°C (40x);
10 min 72°C
[7]
40-70
8%
A189f#
Particulate
682r
methane
mb661_nd monooxygenase
(pmoA)
[Methane
oxidation]
GGN GAC TGG GAC TTC TGG
GAA SGC NGA GAA GAA SGC
CCG GCG CAA CGT CCT TAC C
95°C 10 min; 45 s 95°C,
45s 61°C, 1min 72°C (35x);
10 min 72°C
[12, 13]
35-65
7%
4
5
6
7
8
9
10
11
12
13
14
* GC clamp: CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC G
**GC clamp: GGC GGC GCG CCG CCC GCC CCG CCC CCG TCG CCC G
# semi-nested approach, first with Me1f and Me2r, second with Me2r and Me3f-gc, pmoA: first 189f and 682r,
second 189f and mb661_nd
1
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
References:
1. Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by
denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding
for 16S rRNA. Appl Environ Microbiol 59:695–700.
2. Teske A, Sigalevich P, Cohen Y, Muyzer G (1996) Molecular identification of bacteria from a coculture
by denaturing gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in
pure cultures. Appl Environ Microbiol 62:4210–4215.
3. Lee SH, Malone C, Kemp PF (1993) Use of multiple 16S ribosomal RNA-targeted fluorescent probes to
increase signal strength and measure celluar RNA from natural planktonic bacteria. Mar Ecol Prog Ser
101:193–201. doi: 10.3354/meps101193
4. Hales B, Edwards C, Ritchie D, et al. (1996) Isolation and identification of methanogen-specific DNA
from blanket bog peat by PCR amplification and sequence analysis. Appl Env Microbiol 62:668–675.
5. Nunoura T, Oida H, Miyazaki J, et al. (2008) Quantification of mcrA by fluorescent PCR in methanogenic
and methanotrophic microbial communities. FEMS Microbiol Ecol 64:240–247.
6. Throbäck IN, 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. Holmes AJ, Costello A, Lidstrom ME, Murrell JC (1995) Evidence that participate methane
monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett
132:203–208.
8. Holmes AJ, Costello A, Lidstrom ME, Murrell JC (1995) Evidence that participate methane
monooxygenase and ammonia monooxygenase may be evolutionarily related. FEMS Microbiol Lett
132:203–208.
9. Lin J-L, Joye SB, Scholten JCM, et al. (2005) Analysis of methane monooxygenase genes in Mono Lake
suggests that increased methane oxidation activity may correlate with a change in methanotroph
community structure. Appl Environ Microbiol 71:6458–6462.
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