Ab 3500 genetic analyzer

DNA isolation and amplification were performed following standard protocols in our laboratory (Premachandra et al., 2017a (link)). A panel of eight microsatellite markers were used to genotype the experimental fish (Whatmore et al., 2013 (link); Knibb et al., 2016 (link)). PCR products were separated by capillary electrophoresis on an AB 3500 Genetic Analyzer (Applied Biosystems). Fragment sizes were determined relative to an internal lane standard (GS-600 LIZ; Applied Biosystems) using GENEMARKER v1.95 software (SoftGenetics, State College, PA, United States) and double-checked manually. Parentage assignment was completed using COLONY software (Jones and Wang, 2010 (link)) with confidence scores of above 95%. The pedigree included 65 full-sib groups (16 dams and 31 sires), with the family size of 3–108 offspring. A total of 1,957 offspring out of 1,998 individuals were assigned to full- and half-sib families (Premachandra et al., 2017b (link)). In this study, representatives of large size families were sent to DArT Ptd. Ltd. in Canberra, Australia for sequencing. Seven hundred and fifty-two individual fish sequenced by DArT-seq technology platform contained 35 (full and half-sib) families varying in size between 2 and 39 (average family size = 17 fish).

pMK-T encoding mazF_NE1181 and pET21c were digested with XhoI and BamHI (Toyobo, Osaka, Japan). These linearized DNA fragments were cleaned using a MinElute PCR purification kit (Qiagen, Hilden, Germany). The mazF_NE1181 fragment was then cloned into the multiple cloning sites of pET21c using a DNA ligation kit (Takara), generating the plasmid pET21c-mazF_NE1181. E. coli strain DH5α (Nippon Gene, Tokyo, Japan) was transformed with pET21c-mazF_NE1181, and this transformant was grown at 37 °C on LB plate containing 100 μg/mL ampicillin. pET21c-mazF_NE1181 was extracted using the QIAprep Spin Miniprep Kit (Qiagen), and the sequence was confirmed using an AB 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.

Double‐stranded DNA fragment encoding mazF_DR0417 gene was digested with EcoRI and BamHI (Toyobo, Osaka, Japan) and purified using a MinElute PCR purification kit (Qiagen, Hilden, Germany). Likewise, the pMX‐T plasmid encoding D. radiodurans mazEF genes (mazEF_DR0416DR0417) was digested with BamHI (New England Biolabs, Ipswich, MA, USA) and cleaned with a QIAquick PCR purification kit (Qiagen). The linearized pMX‐T plasmid was then digested with EcoRI (Takara), and the DNA fragment containing mazEF_DR0416DR0417 genes was recovered by using a QIAquick gel extraction kit (Qiagen). These DNA fragments were then ligated into the corresponding pET21c multiple cloning site using a DNA ligation kit (Takara), producing pET21c‐mazF_DR0417 and pET21c‐mazEF_DR0416DR0417, and they were introduced into the E. coli strain DH5α (Nippon Gene, Tokyo, Japan). pET21c‐mazF_DR0417 and pET21c‐mazEF_DR0416DR0417 were extracted using a QIAprep Spin Miniprep Kit (Qiagen) and the sequences were validated using an AB 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's protocol.

PCR was carried out to amplify the antitoxin (mazEpp) and the toxin (mazFpp) genes with Taq HS DNA polymerase (Takara) using genomic DNA of Pseudomonas putida as a template. These PCR products were purified by a PCR purification kit (Qiagen, Venlo, Netherlands). pET21c and PCR products were digested with BamHI and EcoRI (Toyobo, Osaka, Japan) and cleaned using phenol/chloroform extraction and ethanol precipitation. Digested mazEpp and mazFpp fragments were inserted into the corresponding pET21c multiple cloning sites with a DNA ligation kit (Takara). This generated the plasmids pET21c-mazEpp and pET21c-mazFpp, respectively. Each plasmid was then introduced into Escherichia coli DH5α and the colonies were grown overnight at 37°C on LB plate containing ampicillin (100 μg/ml). Plasmids were extracted using QIAprep Spin Miniprep Kit (Qiagen) and their sequences were determined using AB 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.

All necessary Good Lab Practices (GLP) were employed to avoid artifacts. The precautions were taken to avoid the contamination of serum samples by bar coding of samples. The cross contamination was prevented by setting of the PCR independently in the separate tubes with pair of specific primers. The tube containing no template was also setup as a negative control. The RT-PCR product (511 bp) obtained using D1 and D2 primers was separated on the 1% agarose gel. The DNA band was eluted from the gel using Wizard gel and PCR clean up system (Promega, Madison, WI, USA) as per the manufacturer instructions and was sequenced directly using big dye terminator V3.1 ready reaction sequencing mixture in an automated AB3500 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Additionally, nested PCR products obtained using D1 and TS1/TS3 dengue virus serotype specific primers were purified and sequenced as detailed above.

To eliminate contamination as a confounding factor, we performed STR analysis of all CTC samples and compared it to that of the primary tumor and whole blood with the GenePrint 10 v1.1 system (Promega, Madison, WI) using the manufacturer’s recommended protocol: a 10 ng aliquot of template DNA was added to the amplification master mix and amplified for 30 cycles on a GeneAmp PCR System 9700 thermal cycler (Thermo). Fragments were analyzed on a AB 3500 Genetic Analyzer with POP-4 Polymer (Applied Biosystems, Foster City, CA) and visualized using GeneMapper 5 software (Applied Biosystems).