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 .
