Abi prism bigdye terminator version 3

Viral DNA extraction from fecal suspensions for PCR and genotyping was performed using the QIAamp DNA mini Kit (Qiagen, Hilden, Germany) and the QIAcube platform (Qiagen). AdV hexon genotyping was performed by PCR and sequencing using a specified primer set (ADHEX1F/AD2) according to previous studies with few modifications [11 (link), 12 (link)]. For DNA extracts that could not be amplified by this primer set, a different primer set (AD1/AD2) was used for PCR [12 (link)]. The PCR products were visualized by electrophoresis on an agarose gel and analyzed by DNA sequencing. The nucleotide sequences were analyzed using ABI Prism BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA), and genotypes were confirmed using the NCBI BLAST server of the GenBank database.

KRAS mutation tests were performed at the designated central laboratory of SMC as described previously [31 (link)]. Mutations in codons 12 and 13 of the KRAS gene were detected by direct sequencing of polymerase chain reaction (PCR) products amplified from DNA extracted from representative tumor tissue. BRAF V600E direct sequencing and KIT hotspot mutations were tested according to our previous work [32 (link)]. Briefly, tumor-rich areas (>80%) were extracted from paraffin–embedded tissue sections, and 10 4-μm-thick sections containing a representative portion of each tumor block were subjected to DNA isolation using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Deeply pigmented samples were incubated with Chelex-100 (Bio-Rad Laboratories) to prevent PCR inhibition by melanin [33 (link)]. Purified DNA was incubated for 10 min at room temperature with an equal volume of a 5% Chelex-100 solution equilibrated in Qiagen AE buffer, heated to 95°C for 2 min, and allowed to cool. The Chelex-100 resin was pelleted in a microfuge, and the supernatant DNA used for PCR reactions. PCR products were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster, CA, USA) with both forward and reverse sequence-specific primers. Sequence data were generated using the ABI PRISM 3100 DNA Analyzer (Applied Biosystems).

For DNA extraction, a bacterial suspension was prepared in 500 μl of 0.2 mM EDTA, 30 μl of 1 M NaOH was added, and the sample was boiled for 5 min. After 2 min centrifugation at 16.000 x g, the supernatant was recovered. PCR amplification was performed with a DNA thermocycler (Eppendorf). Each reaction mixture contained 5 μl of PCR buffer (EG Healthcare) and 5 μl of each of the four deoxynucleoside triphosphates (Roche) at a final concentration of 200 μM each. A total of 2.5 μl of each primer was used at a concentration of 10 μM, with 5 U of Taq DNA polymerase (EG Healthcare), in a total volume of 50 μl. The cycling conditions for the rpoD (PsEG30F/PsEG790R) [18 (link)], 16S rRNA (16F27/16R1492) [23 ] and gyrB (BAUP2/APrU) genes [24 (link)] included a denaturation period at 94°C for 5 min, followed by 30 cycles of amplification (denaturation at 94°C for 1 min, primer annealing at 55°C (48°C for rpoD) for 1 min, and primer extension at 72°C for 1.5 min). A final elongation step was carried out at 72°C for 10 min. The amplified products were purified with MultiScreen HTS PCR 96-well filter plates (Millipore) according to the manufacturer’s instructions. Sequencing reactions were performed using ABI Prism BigDye Terminator version 3.1, and the sequences were read with an automatic sequence analyzer (3130 genetic analyzer; Applied Biosystems).