A degenerate primer reverse transcriptionpolymerase chain reaction protocol to determine the diversity of picorna-like viruses
Whole seawater (if intracellular viruses are to be included in the analysis) or 0.22-filtered seawater (if only the free virus community is targeted) is gently filtered (7 mmHg) through a 0.02 µm aluminum oxide filter (Anotop, Whatman).
Filtration should continue until the rate slows dramatically or drops to zero.
Use pumped air to remove as much residual sample as possible.
Once dry, seal the filter inlets and outlets with parafilm.
Label the filter.
Flash-freeze the filter in liquid nitrogen.
Store it at –80°C until extraction.
Total nucleic acids are extracted from the aluminum oxide filters using a Masterpure complete DNA and RNA Purification kit (Epicenter, Biotechnologies).
Remove contaminating DNA from RNA preparations with the Turbo DNA-free kit (Applied Biosystems) as described in the protocol provided with the kit.
Each cDNA reaction contains 11 µL of extracted Turbo DNA-free–treated RNA template, 0.2 mM of each dNTP, and 100 ng of N6 primer in a total volume of 13 µL.
Denaturation and annealing of the primers to the RNA template occurs by heating the sample to 65°C for 5 min and then cooling it on ice.
While still on ice, DTT (0.5 mM final conc.) is added to the reaction as an enzyme stabilization reagent with 40 U RNase OUT (Invitrogen) to protect the sample from RNAse activity.
The complementary DNA strand is synthesized with 200 U Superscript III (Invitrogen) Reverse Transcriptase.
The final reaction volume should be 20 µL.
The reaction is incubated initially at 25°C for 5 min so that the relatively unstable hexamer primers remain annealed to the template while cDNA synthesis commences.
The temperature is then increased to 50°C, the temperature at which Superscript III’s processivity is highest, for 60 min.
The activity of the enzyme is terminated by incubating the reaction at 85°C for 5 min.
After cDNA synthesis, add 1 µL (2 U) of RNase H (Invitrogen) to the reaction and incubate at 37°C for 20 min to digest the RNA template from the cDNA:RNA molecule.
PCR can be performed with the RdRp, Mpl.sc1, Mpl.sc2, Mplsc3, and Mpl.cdh primer sets listed in Table 1 and the cDNA synthesized in the previous step.
In a 0.2 mL nuclease-free PCR tube, add reaction components to achieve final concentrations of 1× Platinum Taq buffer, 3 mM MgCl2, 0.2 mM of each dNTP, 1 µM of each primer, and 1 unit of Platinum Taq DNA polymerase (Invitrogen).
Incubate the reactions with the following thermal cycling conditions: 94°C for 75 s (this is necessary to activate the enzyme and ensures complete initial denaturation of template), followed by 35 cycles of denaturation at 94°C for 45 s, annealing at a primer-specific temperature (Table 1) for 45 s, and extension at 72°C for 45 s. 
Complete the cycle with a single final extension step of 9 min 15 s at 72°C.
Before gel separation, we purify and concentrate the PCR reactions with a MinElute PCR cleanup column (Qiagen) as described by the manufacturer.
Purified PCR products are loaded onto a 1% agarose gel containing 1× SYBR safe stain (Invitrogen) and 0.5× TBE buffer.
Bands of DNA of the appropriate size (approximately 500 bp) are excised and purified with a MinElute Gel Extraction kit (Qiagen) according to the manufacturer’s instructions.
We recommend eluting DNA from the column with three washes of 10 µL nuclease-free water in preparation for the end repair reaction.
In preparation for ligation, PCR products are polished and phosphorylated with the PCRTerminator End Repair kit (Lucigen) as described by the manufacturer.
After a 15-min incubation at room temperature the reaction is purified and concentrated with a MinElute Reaction Cleanup column (Qiagen).
The eluted PCR products are subsequently ligated into the pSMART-HCKan vector (Lucigen) according to the manufacturer.
After terminating the ligation reaction by incubating for 15 min at 70°C, 2 µL of the ligation reaction is transformed into Ecloni 10G Supreme cells (Lucigen) via electroporation.
Before initiating a large-scale sequencing effort, we recommend screening 10 to 20 colonies for inserts by PCR amplification with the primers SL1 and SR2 (Table 1) to assess the quality of the library.
Colony PCR products can be visualized on a gel and the products in the correct size range purified and sequenced.
