2. Methodology
2.2. Nucleic Acid Extraction
2.2.1. Extraction
DNA and RNA were extracted from environmental samples and SIP microcosms using the method outlined in Purdy (2005). This methodology was used as it has been shown to consistently provide high yields and purity of extracted nucleic acids (Purdy, et al., 1996; Purdy, 2005) and allowed for the extraction of either total nucleic acids, only DNA, only RNA or separate DNA and RNA from one environmental sample. This methodology has also been found to be able to effectively extract nucleic acids from environments with low bacterial abundances (Zanardini, et al., Unpublished; and personal communication with E. Monagahan, University of Warwick).
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2.2.1.1. Medway Sediment
Nucleic acids were extracted from 0.5 - 0.7 g of homogenised estuarine sediment. Sediment was weighed in autoclaved 2 ml screw capped microcentrifuge tubes, with 0.5 g of DNA/RNA free glass beads, using a clean, sterile spatula and extracted as outlined by Purdy (2005).
2.2.1.2. ETNP Samples
For marine substrates, nucleic acids were extracted from either half of or a full polycarbonate filter. Due to low bacterial abundances from marine SIP incubations it was necessary to use the whole filter during extraction in order to obtain suitable quantities of DNA for downstream analyses. Filters were removed from storage and cut in half using a sterile scalpel and forceps. Cut filters were directly inserted into autoclaved 2 ml screw-capped microcentrifuge tubes with 0.5 g of DNA/RNA free glass beads and lysis buffer (see Purdy, 2005). DNA was then extracted as outlined in (Purdy, 2005). Using this method, the entire filter was seen to disintegrate/dissolve during lysis, thus negating the need to scrape, or wash cells from the filter as described in other extraction methods from aquatic samples.
2.2.2. Purification
It was occasionally necessary to perform a further purification step before sample analysis and downstream processing. This was particularly needed whilst processing sediment samples as it was discovered, even after extraction, that there were still large quantities of impurities (e.g. humic compounds) which hindered further processing (e.g. PCR).
DNA was purified using a Poly-ethylene-glycol (PEG) precipitation method adapted from Selenska and Klingmuller (1991) and Arbeli and Fuentes (2007). 0.2 volumes of a 5M NaCl solution and 1 volume of a 30% PEG6000 (Sigma-Aldrich) solution
was added to DNA samples to be purified. Samples were incubated at 4°C overnight and then centrifuged for 30 minutes at 13,000 rpm. The resulting pellet was then washed twice with a 70% ethanol solution, again centrifuging for 30 minutes at 13,000 rpm between each wash. The supernatant was removed and the pellet left to
28 dry naturally for 15-30 minutes. The pellet was then re-suspended in 30-50 µl of a 10 mM Tris pH 7.5 solution.
2.2.3. Quantification
Extracted nucleic acids were quantified either by agarose gel electrophoresis with comparison against commercial ladders (Bioline, UK and Invitrogen, UK) or using a Nanodrop ND-1000 Spectrophotometer.
Typically 5 μl of aqueous, nucleic acid solution was mixed via gentle pipetting with 2 μl of loading dye and loaded onto 1% agorose gels (Bio-rad, UK). Agarose gels were run for between 30 minutes to 1 hour with a constant voltage of ~5 V cm-1 in 1x TAE buffer.
Aqueous nucleic acid samples were analysed via a Nanodrop ND-1000 Spectrophotometer as per the instructions which came with the instrument.
2.3. Environmental Analysis
2.3.1. Overview
The diversity and distribution of anammox organisms across environmental gradients was investigated using both a phylogenetic (16S rRNA) and functional (hzo) approach. Due to the difficulties associated with investigating anammox ecology (e.g. low abundance) it was necessary to develop a suite of PCR primers which would be able to effectively isolate and amplify target anammox sequences from the environment.
2.3.2. PCR
2.3.2.1. 16S rRNA
2.3.2.1.1. Primers
A range of PCR primers targeting anammox 16S rRNA genes were utilised during this project (Table 2.1). A full description of the primers used to detect anammox 16S rRNA genes during this study and their validation is given in chapter 3. PCR reaction protocols for each set of primers can be seen in Table 2.1.
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Table 2.1: Table showing list of 16S rRNA primers used in this study.
Primer Name Primer Sequence Specificity
Annealing Temperature (°C) Approximate Amplicon Length (bp) Reference 16S 1
Pla46F 5’-GGA TTA GGC ATG CAA GTC-3’ Planctomycetales
62 1344
(Neef et al., 1998)
1390R 5'-GAC GGG CGG TGT GTA CAA-3' Bacteria (Zheng et al.,
1996)
16S 2
Amx368F 5’-TTC GCA ATG CCC GAA AGG-3’ All Anammox
62 480
(Schmid et
al., 2003)
Amx820R 5’-AAA ACC CCT CTA CTT AGT GCC C-3’ Non-Scalindua Anammox (Schmid et
al., 2000)
16S 3
Amx368F 5’-TTC GCA ATG CCC GAA AGG-3’ All Anammox
62 480
(Schmid et al., 2003)
BS820R 5’ -TAA TTC CCT CTA CTT AGT GCC C-3’ Ca. Scalindua spp. (Kuypers et
al., 2003)
16S 4
Amx368F-GC 5’-CCG CCG CGC GGC GGG CGG GGC GGG GGC ACG GGG TTC GCA ATG CCC GAA AGG-3’ All Anammox
62 510
This Study
Amx820R 5’-AAA ACC CCT CTA CTT AGT GCC C-3’ Non-Scalindua Anammox (Schmid et
al., 2000)
16S 5 An7F 5'-GGC ATG CAA GTC GAA CGA GG-3' All Anammox 63 1380 (Penton et al., 2006)
An1388R 5'- GCT TGA CGG GCG GTG TG-3'
16S 6
An7F 5'-GGC ATG CAA GTC GAA CGA GG-3'
All Anammox 63 511
(Penton et al., 2006)
518R 5'-ATT ACC GCG GCT GCT GG-3' (Muyzer et
al., 1993)
16S 7 Brod541F 5'- GAG CAC GTA GGT GGG TTT GT-3' Ca. Scalindua spp. 60 719 (Penton et al., 2006)
Brod1260R 5'-GGA TTC GCT TCA CCT CTC GG-3'
16S 8 B540F 5'-GCT ACC GAA AGG GTT GCT AA Non-Scalindua Anammox 55 669 This Study
B1209R 5'-CCA TCG TTT ACG GCT AGG AC-3'
16S 9 J697F 5'-AGG GTA AAG GCC TAC CAA GG-3' Ca. Jettenia spp. and Ca.
Kuenenia spp. 58 568 This Study
J1265R 5'-CAA AAC CCC TCT ACC GAG TG-3'
16S 10 K580F 5'-GCA AAA GCA CTT GTG GTC AA-3' Ca.Kuenenia spp. 51 467 This Study
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Table 2.2: Table showing PCR reaction conditions for PCRs targeting anammox 16S rRNA genes. All
PCRs were performed using a hot start at 96°C. Primer set numbers collaborate with those shown in Table 2.1. The initial 10 cycles of PCR reaction 16S 1 utilised a touch-up PCR methodology, rising from 57°C to 62°C in increments of 0.5°C per cycle.
2.3.2.1.2. Promega Reagents
PCR was performed in either sterile 0.2 ml eppendorf tubes or 96 well PCR plates (Thermo-Scientific, UK). PCR was performed in 50 μl reactions using reagents and DNA Go-Taq polymerase (Promega, UK), dNTPs (Invitrogen, UK) and sterile, nuclease free water (Ambion, UK). The concentrations and volumes of PCR reagents are shown in Table 2.3. PCR was performed in an Eppendorf Mastercycler epgradient thermocycler using the specific conditions outlined in Table 2.2.
Primer
Set Hot Start
No.
Cycles Denaturation Annealing Elongation
Final Elongation
16S 1 95 °C for 2 min a) 10 95 °C for 45 s 57 °C - 62 °C for 50 s
* 72 °C for 1 min 22 s 72 °C for 5 min b) 20 62 °C for 50 s
16S 2 95 °C for 2 min 30 95 °C for 45 s 62 °C for 50 s 72 °C for 1 min 22 s 72 °C for 5 min
16S 3 95 °C for 2
min 30 95 °C for 45 s 62 °C for 50 s
72 °C for 1 min 22 s
72 °C for 5 min
16S 4 95 °C for 2
min 30 95 °C for 45 s 62 °C for 50 s
72 °C for 1 min 22 s
72 °C for 5 min
16S 5 96 °C for 2
min 30 96 °C for 45 s 63 °C for 1 min 72 °C for 1 min
72 °C for 7 min 16S 6 96 °C for 2 min a) 10 96 °C for 1 min 63 °C for 30 s 72 °C for 2 min 30 s 72 °C for 10 min
b) 20 94 °C for 30 s 72 °C for 2 min
16S 7 96 °C for 2
min 30 96 °C for 45 s 60 °C for 1 min 72 °C for 1 min
72 °C for 7 min 16S 8 96 °C for 2 min a) 10 96 °C for 1 min 55 °C for 30 s 72 °C for 2 min 30 s 72 °C for 10 min
b) 20 94 °C for 30 s 72 °C for 2 min
16S 9 96 °C for 2 min a) 10 96 °C for 1 min 58 °C for 30 s 72 °C for 2 min 30 s 72 °C for 10 min
b) 20 94 °C for 30 s 72 °C for 2 min
16S 10 96 °C for 2 min a) 10 96 °C for 1 min 55 °C for 30 s 72 °C for 2 min 30 s 72 °C for 10 min
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Table 2.3: Table showing concentrations and volumes of PCR reagents for PCR carried out with Promega
reagents. Volumes are for one reaction.
2.3.2.1.3. Bioline Reagents
PCR was also performed using reagents produced by Bioline, UK. One reaction contained: 12.5 μl Bioline My-Taq Red master mix, 1 μl of 10 μM of forward primer solution, 1 μl of 10 μM of reverse primer solution, 9.5 μl of sterile, nuclease free H2O and 1μl of DNA solution to a final volume of 25 μl. PCRs were performed in an
Eppendorf Mastercycler epgradient thermocycler using the specific conditions outlined in Table 2.2.
2.3.2.2. hzo Functional Genes
2.3.2.2.1. Primers
A range of primers targeting anammox hzo were used in this project (Table 2.4). A full discussion of these primers can be read in chapter 4.
Table 2.4: Table showing PCR primer sequences used during the investigations into the use of hzo as a molecular marker of anammox.
Reagent Concentration Volume Final Concentration
PCR Reaction Buffer 5X 10 μl 1X MgCl2 25 mM 4 μl 2 mM dNTPs 25 mM (each) 0.5 μl 0.25 mM (each) Forward Primer 10 μM 2 μl 0.4 μM Reverse Primer 10 μM 2 μl 0.4 μM Bovine Serum Albumin (BSA) 100 μg/ml 2 μl 4 μg/ml
Taq DNA Polymerase 5 Units/μl 0.2 μl 1 Unit (0.02 Units/μl)
DNA Variable 1 μl -
ddH2O Pure Up to 50 μl (28.3 μl) -
Primer Set
Primer
Name Primer Sequence
Length
(bp) Reference hzo 1 hzocl1F1 5’-TGY AAG ACY TGY CAY TGG-3’ 470 (Schmid et al., 2008)
hzocl1R2 5’-ACT CCA GAT RTG CTG ACC-3’
hzo 2 hzocl1F1l 5’-TGY AAG ACY TGY CAY TGG G-3’ 471 (Schmid et al., 2008)
hzocl1R2 5’-ACT CCA GAT RTG CTG ACC-3’
hzo 3 HZO4F 5’-TTG ART GTG CAT GGT CTA WTG AAA G-3’ 1037 (Hirsch et al., 2011)
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2.3.2.2.2. Protocol
PCR was performed with primers targeting hzo as outlined in section 2.3.2.2 and Table 2.3 except that, for primers hzocl1F1 & hzocl1R2 and hzocl1F1L & hzocl1R2 (hzo 1 and hzo 2 in tables), 2.5 μl of 25 mM MgCl2 (c.f. Table 2.3) was added per
reaction to a final concentration of 1.25 mM as performed by Schmid, et al. (2008). PCR was performed in an Eppendorf Mastercycler epgradient thermocycler using the conditions outlined in Table 2.5.
Table 2.5: Table showing PCR protocols used with primers targeting anammox hzo. All PCRs were
initiated with a hot start at 94 °C. Primer set numbers collaborate with those in Table 2.4.
2.3.2.3. Analysis
The presence or absence of amplifiable product was determined via electrophoresis on 1% agarose gels as described in section 2.2.3. PCR product was quantified either by visual comparison of agarose gels or using a Nanodrop ND-1000 Spectrophotometer (section 2.2.3).
2.3.2.4. PCR Purification
PCR products required for downstream processing (e.g. sequencing) were purified to remove impurities which may remain after PCR (e.g. unbound dNTPs). PCR products were purified either using a PCR Purification Kit (Qiagen, UK) or were extracted from 1% agarose gels using a Gel Extraction Kit (Qiagen, UK).