Automated dna sequencer

Each amplified PCR product was purified from agarose gels using the QIAquick Gel Extraction Kit (Qiagen GmbH, Hilden, Germany) and by QIAquick PCR Purification Kit (Qiagen). The sequencing of purified PCR products was performed using an automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) using the forward and reverse primers used in the Vibrio multiplex PCR. The sequencing data was compared with the known targeted gene sequences which were originally used for specific-primer design for each Vibrio species.

A viral kit for DNA extraction (Promega Maxwell) was used. β-globin polymerase chain reaction (PCR) with the primers PCO3 and PCO4 was carried out on all samples to ensure the presence of human DNA and that no PCR-inhibiting agents were present.^{27 (link)} All samples were analyzed with a nested PCR system (MY09/11) and GP^5+/6+ that detects most mucosal HPV types and all high-risk HPV types that have oncogenic potential in mucosal tissue.^{28 ,29 (link)} All HPV-positive DNA samples were sequenced to confirm viral DNA sequences. For sequencing, HPV-positive PCR products were purified with a PCR purification kit in a magnetic 96-ring SPRIplate (Agencourt Biosciences). Sequencing reactions were performed containing the purified PCR products together with GP⁺ primer and BigDye Terminator. Sequence reactions were purified with a dye-terminator removal kit (Agencourt Biosciences) in a magnetic 96-ring SPRIplate. Direct sequencing was conducted, and sequence reactions were analyzed with an automated DNA sequencer (Applied Biosystems). The DNA sequences were compared with available sequences in GenBank through the National Center for Biotechnology Information BLASTn suite server.³⁰ Participants with β-globin–positive saliva samples were included in the data analysis. (β-globin is a DNA integrity check; any samples with negative β-globin were invalid.)

5′ and 3′ RACE analyses were performed using the GeneRacer kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. Total RNA was extracted from three midguts of day three of the fifth instar silkworm larvae using Trizol reagent (Invitrogen, Carlsbad, CA, USA), followed by treatment of RNase-free DNase I (Promega, Fitchburg, WI, USA) for 30 min at 37 °C to eliminate the contaminating genomic DNA. The purity of extracted RNA was determined by UV spectrophotometer. Four μg of RNA was reverse-transcribed to the first strand of cDNA using M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) for 1 h at 42 °C. In order to obtain the full-length cDNA of the BmCDA7, specific primers were designed by primer 5 (Table S4), and then the procedure was performed using the GeneRacer kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. All the PCR products were electrophoresed on 2% agarose gels containing ethidium bromide and photographed under UV illumination. cDNA fragments were extracted from agarose gels, purified using a agarose gel purification kit (Axygen, Union City, CA, USA), and cloned to pEASY-T1 simple vector (TransGen, Beijing, China). The cloned product was sequenced using automated DNA sequencer (Applied Biosystems 3730, Shanghai, China).