Validating Segmental Trisomy Genotyping by Breakpoint PCR

Primers spanning the translocation breakpoint were designed using Primer3 software (Broad Institute). The breakpoint primer sequences were Chr17fwd_5′-GTGGCAAGAGACTCAAATTCAAC-3′ and Chr16rev_5′-TGGCTTATTATTATCAGGGCATTT-3′. These primers amplify a ~275 bp product from junction site of the 17¹⁶ translocation product. Positive control primers were IMR8545_5′-AAAGTCGCTCTGAGTTGTTAT-3′ and IMR8546_5′-GGAGCGGGAGAAATGGATATG-3′, which amplify a 600 bp product from the Rosa locus. In a 50 μl total reaction, all 4 primers were used at a final concentration of 0.4 μM each. PCR cycling conditions were 94°C – 2min, 94°C – 45sec, 55°C – 45sec, 72°C –1min for 40 cycles, followed by a final 7 min. extension at 72°C – 7min. PCR products were separated on a 1.5% agarose gel. For the purposes of validation, 209 samples that were previously genotyped by either by quantitative PCR (Liu et al. 2003 ) or DNA FISH as being either positive, negative or inconclusive for the segmental trisomy were genotyped by breakpoint PCR. Among this set of samples were samples from Ts65Dn mice and 2 derivative strains, B6EiC3Sn.BLiA-Ts(17¹⁶)65Dn/DnJ (The Jackson Laboratory, stock #5252) and B6EiC3Sn-Rb(12.Ts17¹⁶65Dn)2Cje/CjeDn (The Jackson Laboratory, stock #4850). Additionally, 113 samples derived from Ts65Dn mice and genotyped using the SNP based genotyping prescreen (Lorenzi et al 2010) and FISH confirmation (Moore et al. 1999 (link)).