DNA extraction
The Niskin bottles were drained into acid washed polycarbonate bottles, and then the seawater was filtered through sterile polypropylene filter holders using a peristaltic pump. Two litres of seawater from each depth were serially filtered through a 25 mm diameter, 10 um pore-size polyester filter (GE Osmonics, Minnetonka, MN) and a 25 mm, 0.2 um poresize Supor filter (Pall Corporation, Port Washington, NY, USA). Each filter was then stored in a polypropylene microcentrifuge tube, which contained 500 ul of Tris EDTA buffer (Ambion, Foster City, CA, USA) plus an approximate 0.2 g mixture of 0.1 mm and 0.5 mm diameter autoclaved glass beads (BioSpec Products, Bartlesville, OK, USA). The samples were immediately flash frozen in liquid nitrogen and subsequently stored frozen at -80°C until processed for nucleic acid extraction.
DNA was extracted from the samples using the modified DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) protocol detailed in Moisander and colleagues (2008). However, during the final elution step, the samples were eluted twice with 25 ul of Buffer AE instead of 100 ul. The extracted DNA was stored at -20°C.
Quantitative PCR
A qPCR method using a TaqMan 5'-fluorogenic exonuclease assay was used to investigate the abundance and distribution of several nifH cyanobacterial sequence types, called nifH phylotypes. Probes were 5' labelled with the fluorescent reporter FAM (6-carboxyfluorescein) and 3' labelled with the quenching dye TAMRA (6-carboxytetramethylrhodamine). TaqMan oligonucleotide primers and specific fluorogenic probes were used to target the nifH gene of three unicellular cyanobacteria (groups A, B and C, also known as UCYN-A, UCYN-B and UCYN-C) and Trichodesmium spp., and the host-specific nifH sequences of three diatom-cyanobiont symbioses (H-R, R-R and C-C) (Table 4) (Church et al., 2005a; Church et al., 2005b; Foster et al., 2007). The cyanobionts associated with each type of diatom have been designated, based of nifH sequences, as het-1 (H-R), het-2 (R-R) and het-3 (C-C). The primer and probe sets for the diatom-cyanobiont symbioses were used to analyse extracts from the > 10 um size fraction of each sample, whereas the primer and probe sets for unicellular cyanobacteria were used to analyse extracts from the < 10 um size fraction (0.2 um to 10 um). The Trichodesmium primer and probe sets were run on extracts from both size fractions of each sample.
The qPCR reactions were prepared in 96-well optical reaction plates with optical caps (Applied Biosystems, Foster City, CA, USA) and run on a ABI 7500 Real-time PCR System (Applied Biosystems) with the following thermocycling settings: 50°C for 2 min, 95°C for 10 min, and then 45 cycles of 95°C for 15 s followed by 60°C for 1 min. The sample reactions (25 ul) were run in triplicate and contained 12.5 ul of 2 x TaqMan Mastermix (Applied Biosystems), 8 ul of 5 kD filtered nuclease-free water (Ambion), 1 ull each of the forward and reverse primers (0.4 uM final concentration), 0.5 ul of fluorogenic TaqMan probe (0.2 uM final concentration) and 2 ul of template DNA (ranging from 0.05 to 161.27 ng DNA). Controls without any DNA target (no template controls) contained 2 ul of 5 kD filtered nuclease-free water (Ambion) instead of the template DNA, and were run on each plate to check for contamination.
Linearized recombinant plasmids containing the nifH gene targets were used as standards for qPCR by making a dilution series covering 8 orders of magnitude (100–107 nifH gene copies per reaction), and the full series was run on each plate. The nifH gene copies l-1 were calculated for each nifH target set using linear regression parameters fit to a plot of cycle threshold (Ct) versus log gene copy for the standards run on each plate. The average efficiencies of the qPCR reactions for each of the assays were 102 ± 2% for UCYN-A, 102 ± 3% for UCYN-B, 98 ± 3% for Trichodesmium sp., 93 ± 3% for H-R and 101 ± 3% for R-R.
Each sample was tested for inhibition using one primer/ probe set by spiking the qPCR reaction with a standard of 104 nifH gene copies per reaction, and determining the per cent inhibition using the following formula 1 - [(Ct,sample - Ct,standard)/ Ct,standard] x 100. In the few cases (less than 2% of the samples) where inhibition was observed, the sample was serially diluted until the addition of template DNA did not cause inhibition. In a majority of these cases, the extent of inhibition was minor, as non-inhibited amplification was observed after 10-fold dilutions.
The LOD and limit of quantification (LOQ) have been determined empirically to be 1 and 8 nifH gene copies per reaction, respectively (data not shown). Taking into consideration the qPCR reaction volume, volume of nucleic acid extractions, and the volume of seawater filtered, this translates to a LOD of 10 nifH gene copies l-1 seawater and LOQ of 80 nifH gene copies l-1 seawater for a majority of the samples. LODs and LOQs are considerably higher for samples that needed to be diluted. Samples where amplification was observed, but the detected signal fell below the LOQ, were designated as ‘detected not quantified’ (DNQ). In order to calculate the depth-integrated nifH gene copies m-2, a sample that was below the LOD was considered to have zero nifH gene copies l-1, and a DNQ sample was assumed to have one nifH gene copy l-1.
Table 4. Oligonucleotide TaqMan primers and probes used to detect the nifH phylotypes, and the corresponding target base region.
Target Forward primer (5'-3') Probe Reverse primer (5'-3')
UCYN-Aa AGCTATAACAACGTTTTATGCGTTGA TCTGGTGGTCCTGAGCCTGGA ACCACGACCAGCACATCCA
106–131 133–153 1 56–174
UCYN-Ba TGGTCCTGAGCCTGGAGTTG TGTGCTGGTCGTGGTAT TCTTCTAGGAAGTTGATGGAGGTGAT
138–157 160–176 178–203
UCYN-Cb ATACCAAGGAATCAAGTGTGTTGAGT CGGTGGTCCCGAGCCTGGAG ACCACGACCAGCACATCCA
106–124 133–153 156–174
H-Rb TGGTTACCGTGATGTACGTT TCTGGTGGTCCTGAGCCTGGTGT AATGCCGCGACCAGCACAAC
106–124 133–155 158–177
R-Ra CGGTTTCCGTGGTGTACGTT TCCGGTGGTCCTGAGCCTGGTGT AATACCACGACCCGCACAAC
105–124 133–155 158–177
C-Cb CGGTTTCCGTGGCGTACGTT TCTGGTGGTCCAGAACCTGGTGT AATACCACGACCAGCACAAC
106–124 133–155 133–155
Trichodesmiuma GACGAAGTATTGAAGCCAGGTTTC CATTAAGTGTGTTGAATCTGGTGG CGGCCAGCGCAACCTA
TCCTGAGC
217–241 246–278 284–300
The probes were 5' labelled with the fluorescent reporter FAM and 3' labelled with the quenching dye TAMRA.
a. Primer and probe designed by Church et al., 2005a.
b. Primer and probe designed by Foster et al., 2007.
BCO-DMO Processing Notes
Generated from original spreadsheet contributed by Kendra Turk
"Diazo_Distribution_Zehr_forBCO-DMO.xlsx", tab: SJ0609 nifH qPCR DNA
BCO-DMO Edits
- CRUISE (cruise id) inserted
- Parameter names modified to conform to BCO-DMO convention
- "nd" (BCO-DMO flag for no data) inserted into blank fields
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