Nucleic Acid Amplification, Modification, and Sequencing

Primers, probes, and other oligonucleotides, as well as other materials used for nucleic acid amplification, modification and sequencing are shown in section 7.5 in Table 19.

6.5.1 Amplification of VP1 Gene by Reverse Transcriptase PCR

A clinical stool sample positive for NoV genogroup II was partially purified by chloroform extraction, and viral RNA was extracted (see section 6.1.2). The VP1 gene was amplified from genomic RNA by reverse transcriptase (RT)-PCR in a nested two-step reaction using two different sets of primers as follows. The first set of primers consisted of GSK G2 forward primer and ORF 3 reversed primer, both previously described^{111, 237}. This primer set was applied to generated cDNA of NoV’s ORF 2 and 3 using Superscript II RT as per manufacturer’s instructions. Briefly, the primers (2 pmol/reaction each), dNTPs ( 1 µL of dNTP mix containing 10mM each nucleotide), nuclease free water and template RNA were combined and the mixture incubated at 65°C for 5 min and immediately chilled on ice for 2 min. Keeping the reaction on ice, 5x First-strand Buffer (5xFSB), RNAsin (20 U/µL), DTT (0.1 M) and Superscript-Reverse Transcriptase II (2 U/µL) were combined and added to the mixture. The RT-reaction was incubated for 50 min at 42°C followed by an enzyme inactivation step at 70°C for 15 min. An RNAse H treatment was completed subsequent to the inactivation of the RT-enzyme adding 1 µL (2U) for 20 min at 37 C in a water bath.

Of the RT- reaction, 20 % (4 µL) was used as template to amplify the VP1 gene using Platinum Taq® polymerase and previously described VLP primers¹⁹⁵ (cycling conditions: 94°C for 180 s, 35 cycles of: 94°C for 30 s, 66.6°C for 30 s, 68°C for 90 s, final extension: 72°C 240 s). The master mix pipetting scheme is shown in the annex (Table 33).

6.5.2 Amplification of P-domain Gene by PCR

The P-domain gene was amplified from the recombinant VP1 gene, using DNA polymerase Platinum Pfx® polymerase to generate a blunt end PCR product (cycling conditions: 94°C for 300 s, 35 cycles of: 94°C for 30 s, 60.0°C for 30 s, 68°C for 90 s, final extension: 72°C 420 s) with previously described P-domain primers¹⁹⁵. The master mix pipetting scheme is shown in the annex (Table 34).

The VP1 gene which served as template for the P-domain PCR, was obtained by double restriction digestion of the recombinant TOPO plasmid using Eco RI and Bam HI restriction enzymes, as the two restriction sites were included in the primer sites. The restriction digestion was completed as per recommendations of new England Biolabs (NEB), using the NEB 2.1 buffer. The resulting DNA fragment was purified by agarose gel electrophoresis and subsequently extracted from the agarose gel.

95

6.5.3 Amplification of Nucleic Acid Library During SELEX

The 40 nt random DNA library was flanked by primer annealing sites at either side of the random region. Primers for library amplification were used as previously described²³⁸, with the exception of the reverse primer, which was not biotinylated but phosphorylated to enable post PCR complementary strand separation by exonuclease digestion.

To allow amplification of nucleic acid molecules with complex structures, Q-Solution^TM provided through Qiagen One-step RT-PCR Kit™ was added to the master mix as instructed by the manufacturer. DNA amplification was optimized regarding magnesium, primer and polymerase concentration. The optimal master mix pipetting scheme is shown in the annex (Table 35). The DNA library amplification was optimized and amplified with cycling conditions of 94 °C for 3 min, followed 9-25 cycles of: 94°C for 15 s, 55 °C for 10 s and 72°C for 15 s, without final extension step.

6.5.4 Multiplex Reverse Transcriptase Real Time PCR Assay for Norovirus

Norovirus RNA was detected by a RT-qPCR assay as previously described¹¹³. Briefly, the assay is designed as a multiplex assay, detecting NoV GI and GII simultaneously using degenerated cog primers for GI and GII¹¹⁷. Two Cy5 probes were used for GI and one Cy3 probe for GII. Additionally, the PCR master mix includes primers and one TxRed probe for an internal control referred to as internal amplification control (IAC) primers and probe. The master mix pipetting scheme is shown in the annex (Table 36). The RNA was reverse transcribed and immediately amplified by a hot start DNA polymerase in a one tube reaction with the Qiagen OneStep RT-PCR Kit using optimized cycling conditions (50°C for 50 min and 95°C for 15 min followed by 50 cycles of 95°C for 10 s, 53°C for 25 s, and 62°C for 70 s). Fluorescence was read at the end of the 62°C elongation step. All RT-qPCR reactions were completed using SmartCycler II (Cepheid) using default analysis parameters, except that the manual threshold fluorescence units were set to 10.

6.5.5 Amplification of plasmid DNA from Bacterial Colony by PCR

The colony PCR is an easy analysis tool to screen colonies for successful ligation of the DNA-insert into the vector. For colony PCR, a master mix is prepared using gene specific primers or primers flanking the multiple cloning site on the plasmid. Instead of template DNA, a colony from an agar plate is transferred and suspended into the master mix. In case of a positive ligation and depending on the choice of primers the PCR will result in a DNA fragment of a known size.

Depending on the nature of the plasmid used, these primer regions flanking the multiple cloning site can vary. The primer sets flanking the multiple cloning sites of the different vectors used in this work are named T7 and M13 and the primer sequences are shown in section 7.5 (Table 20).

The pipetting scheme of the master mix is summarized in the annex (Table 35) and the PCR was

96

completed using cycling conditions as per manufacturer’s instructions (cycling conditions: 94°C for 2 min followed by 25 cycles of 94°C for 60s, 55°C for 60 s, and 72°C for 60s, final extension: 72°C 420 s).

6.5.6 Sequencing of VP1 gene PCR product

In this work DNA sequences were obtained using the dye terminator sequencing method, a variant of Sanger Sequencing²³⁹. The VP1 and the P-domain genes were sequenced on the CEQ 8000-Sequencer with the dye terminator cycle sequencing (DTCS) method, using the Genome Lab DTCS Quick Start Kit with optimized cycling conditions ⁽30 cycles of 96°C for 20 s, 50.0°C for 20 s, 60°C for 4 min). For sequencing, primers were designed to anneal to complementary annealing sites located on the VP1 genome to obtain 400 bp fragments (all primers are listed in Table 19), sequencing the 1.6 kb fragment in multiple pieces with subsequent alignment. The primer sites were chosen to produce overlapping fragments, to allow optimal alignment of the resulting sequences. Alignment of the obtained sequences was achieved using the software BioEdit. The sequences were then used to identify the NoV genotype with the database ‘Calicinet’²⁴⁰.

6.5.7 Generation of Single-Stranded Nucleic Acid by Lambda Exonuclease Digestion Lambda exonuclease is an enzyme catalyzing the removal of 5’ mononucleotides from DNA, in this work from PCR product. The preferred substrate of lambda exonuclease is 5’-phophorylated double stranded DNA. The digestion of only the reverse strand of the PCR product, leaving the single-stranded forward strand was accomplished by using a phosphorylated reverse primer during the amplification of the oligonucleotide library.

For exonuclease digestion purified PCR product (see section 6.2.2) amplified with phosphorylated reverse primer was subjected to lambda exonuclease digestion at 37˚C for 50 min with 25 U lambda exonuclease (New England Biolabs, Ipswich MA, USA) in a 50 µL reaction, followed by enzyme inactivation at 70˚C for 10 min. Complete exonuclease digestions was monitored by agarose gel electrophoresis, and single-stranded nucleic acid purified as described in section 6.2.4.

6.5.8 Radionuclide End-Labeling of Oligonucleotides

The 5’-end of DNA-oligonucleotides and PCR products generated with a ‘conventional’, synthesized primer can be directly labeled with γ-phosphate of ATP in a kinase phosphorylation reaction, catalyzed by the polynucleotide kinase (PNK). The direct phosphorylation is possible due to the 5’-hydroxylated-end of synthesized oligonucleotides.

97

Single-stranded nucleic acids were labeled with [γ-³²P]-ATP (Perkin Elmer, Meridan CT, USA) using the Promega T4 Polynucleotide Kinase (Madison WI, USA) according to manufacturer’s instructions. Unincorporated nucleotides were removed as described in section 6.2.3.