Computational protocol: Differential Gene Susceptibility to Sperm DNA Damage: Analysis of Developmental Key Genes in Trout

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[…] The amplification of specific primers in the presumably histone or protamine-bound DNA was assessed by PCR in TGradient Thermocycler (Biometra, Goettingen, Germany). Each reaction (20 µl) contained 200 ng of genomic DNA, 0.25 µM of each primer, 6 mM MgCl2, 800 µM of dNTPs, 1X PCR buffer MgCl2 free (Biotools, Madrid, Spain), 1 U of thermostable DNA polymerase from Thermus sp.(Biotools, Madrid, Spain) and up to 20 µl of bi-distilled water. The annealing temperature was 65°C for long oligonucleotides and the extension time was 30 s. Each product size was confirmed by 1.8% (w/v) agarose gel electrophoresis. [...] Quantitative real-time PCR of genomic DNA was performed in triplicate using specific primers and non-template control for each pair of primers. Two amplicons with different length, located within the same gene are required to determine DNA damage according to the indications of Rothfuss et al., . Oligonucleotide primers were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA). Sequences, corresponding to GenBank accession numbers and PCR efficiency are shown in . Real-time PCR was performed on a Step-One Plus (Applied Biosystems, Foster City, CA, USA) real-time thermal cycler. DNA damage was studied in nine nuclear genes, some of them located in different linkage groups according to Moghadam et al., : HoxA3a-1, HoxB5bi, HoxC4a-2, HoxD4ai, HoxD9aii, Sox2, Eif1b, 18S rRNA (18S Ribosomal RNA), 28S rRNA (28S Ribosomal RNA).Each pair of primers was assayed by conventional and real-time PCR in order to determine optimal conditions for the experiment. Each product size was visualized by agarose gel electrophoresis (data not shown). Reaction conditions were different for long and short fragments, so assays were carried out in different 96-well plates (Applied Biosystems, Spain), using control treatment in each plate. For long amplicons, the reaction mixture contained 4 µl of 5× Fast Start DNA Master plus SYBR Green I (Roche, Germany), 1 µl of each 5 µM forward and reverse primer, 0.4 µl of 50× ROX passive reference dye (BioRad, Foster City), template gDNA (3 ng, except 6 ng for the Sox2 gene assay) and sterile bidistilled water up to 20 µl. In this case, the reaction conditions were a pre-incubation phase of 10 min at 95°C, followed by 50 cycles of 15 s at 95°C, 10 s at the annealing temperature of 65°C (63°C for rRNA 18S) and 50 s at 72°C. For short fragments, the reaction mixture consisted of 10 µl of 2× SYBR Green PCR (Applied Biosystems, Spain), 1 µl of each 5 µM forward and reverse primer, 3 ng of template DNA (6 ng for Sox2) and bidistilled water up to 20 µl. PCR reaction began with a pre-incubation phase of 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at the annealing temperature of 63°C. Product specificity was verified by agarose gel electrophoresis and threshold cycles (Cts) were measured by StepOnePlus version 2.2.2 (Applied Biosystems).Primer efficiencies were determined using serial dilutions of gDNA (up to 1∶1000000 for all oligonucleotides, except for short amplicons corresponding to HoxA3a-1, HoxB5bi, HoxC4a-2 and Sox2 whose serial dilutions were up to 1∶729), corresponding to ∼1000 ng/µl to 0.01 ng/µl of DNA and ∼180 ng/µl to 0.74 ng/µl, respectively. The amplification was made with long and short fragments, employing the same above-mentioned conditions. PCR efficiencies were calculated with StepOnePlus version 2.2.2 (Applied Biosystems) using the linear regression slope of the dilution series (). […]

Pipeline specifications

Software tools Biotools, Primer Express
Applications Population genetic analysis, qPCR
Organisms Danio rerio, Oncorhynchus mykiss
Chemicals Hydrogen Peroxide