Novel genetic and epigenetic alterations in ... - Ous-research.no

specificity, i.e. that they only amplify unique sequences of the human genome, by in silicoPCR, ‡ and for hairpin and/or primer-dimer formation at NetPrimer. §The fragments were labeled with the G5 dye set from Applied Biosystems; PET – red, NED– yellow, VIC – green, and 6-FAM – blue. The size standard used was GS500 LIZ (orange).All fragments were analyzed on a 3730 DNA Analyzer (Applied Biosystems, Foster City,CA, USA) using default Microsatellite Analysis settings in the GeneMapper3.7 software.Electropherograms were visually examined for insertions/deletions by three independentauthors, TA, ECR and MAM, against corresponding fragments from DNA from fourdifferent disease-free individuals. All assays were duplicated in tandem runs using differentPCR machines to ensure the robustness of the results.Mutation verification by sequencingIn order to determine the false discovery rate of the fragment analysis results, 18 of thegenes (ACVR2A, AIM2, ASTE1, AXIN2, BLM, EPHB2, GRK4, MBD4, PTHLH, RAD50,RBBP8, RCC2, SEMG1, SLC23A2, SYCP1, TAF1B, WISP3 and ZMYND8) weresequenced. Genomic DNA from a total of 107 samples was re-amplified with new primersincluding M13-tails, and directly sequenced in both directions on a 3730 Genetic Analyzer(Applied Biosystems). An initial PCR was performed using HotStar HiFidelity Polymerase(QIAGEN) following manufacture’s instructions in a 25μl reaction before enzymatic clean-‡ http://genome.ucsc.edu/cgi-bin/hgpcr§ http://www.premierbiosoft.com/netprimer7