3130 genetic analyzer

The pLV1-puro-DEST vector was prepared as previously described [16 (link),34 (link),59 (link)]. Electrophoretic analysis and DNA sequencing were performed to verify the resulting recombinant vectors. The EGFRwt and EGFRvIII cDNA sequences were obtained, and expression plasmids were created [34 (link),59 (link)]. The coding sequences were transferred to pLV1-puro-DEST under the CMV promoter via the LR reaction (Invitrogen, Waltham, MA, USA). Both sequences were confirmed with an Applied Biosystems 3130 Genetic Analyzer. The transfection was performed with Fugene HD (Promega, Madison, WI, USA) and puromycin (InvivoGen, San Diego, CA, USA), which were used to select cells that had successfully incorporated the plasmid.
Site-directed mutagenesis was performed on pENTR/EGFRvIII as previously described [59 (link)]. After the Gateway LR reaction and incorporation of the mutated EGFRvIII cassettes into pLV-puro-DEST vectors, all sequences were confirmed using the Applied Biosystems 3130 Genetic Analyzer. The primers used for the creation of cysteine–serine EGFRvIII mutants are listed in Table S1A.

Somatic DNA samples were extracted from paraffin–embedded uveal melanoma biopsies with a QIAamp DNA FFPE Tissue Kit (Qiagen Inc., Chatsworth, CA, USA) according to the manufacturer’s instructions. The exon 4 and exon 5 of GNAQ and GNA11 were amplified by nest PCR, which was performed in a Bio–Rad T100 thermal Cycler. Using outer set primers (Table S1) and 10ng extracted somatic DNA as template: 1^st PCR procedure initial denaturation at 95 °C for 3 minutes, followed by 36 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, then extension at 72 °C for 5 min, and keep at 4 °C for 10 min. Using inner set primers (Table S1) and 1 µL 1^st PCR product as template, 2^nd PCR procedure is: initial denaturation at 95 °C for 3 minutes, followed by 33 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s, then extension at 72 °C for 5 min, and keep at 4 °C for 10 min.
Direct nucleotide sequence analysis was completed for both GNAQ and GNA11. The exon 4 and exon 5 of GNAQ and GNA11 were amplified by PCR and sequenced on Genetic Analyzer 3130 (Applied Biosystems). Amplified exon 4 and exon 5 of GNAQ and GNA11 were sequenced on Genetic Analyzer 3130 (Applied Biosystems) using one of inner primers (Table S1).

DNA profiles were generated from a mixture of menstrual fluid and semen. DNA was extracted from (1) the pad underneath the sample well and (2) the remaining sample-buffer solution. For method 1, the plastic cartridge was opened and the pad was cut out and transferred to a reaction tube. For method 2, 100 μl of sample-buffer solution was transferred to a reaction tube. DNA was extracted using the Maxwell® instrument (Promega, Mannheim, Germany) and amplified with PowerPlex® ESX 17 Pro System (Promega) according to manufacturer’s recommendations. Samples were subsequently analyzed using the 3130 Genetic Analyzer with the GeneMapper® ID software by Thermo Fisher Scientific.