Pcr-based pipeline for high-definition dna fish

122 | VOL.10 NO.2 | FEBRUARY 2013 | nature methods probe, which would enable combinatorial labeling and tailoring of the probe size at the discretion of the user, is technically unfeasible with methods based on cloned DNA fragments and is not cost effective with array technology. To overcome these limitations and enable a wider community of researchers to fully exploit the potential of DNA FISH, we designed ready-to-use human and mouse genomic libraries of PCR primer pairs with optimal thermodynamic features, delimiting amplicons 200–220 nucleotides (nt) in length. After filtering out primer pairs that amplify multiple targets and crosshybridizing amplicons, we obtained a database consisting of 4,823,784 and 4,387,601 unique primer pairs for the human and mouse genome, respectively (Fig. 1a). The database can be accessed at www.hdfish.eu. Over 90% of the human and mouse genomes are densely covered by our primers, with more than 80 primer pairs per 100 kb (Fig. 1b,c and Supplementary Fig. 1a,b). Previous attempts to use PCR for unique DNA FISH probe generation were either limited to very few loci or based on time-consuming primer design targeting longer amplicons5,6. In contrast, our primer libraries are easy to access and ready to use, allowing highly specific double-stranded probes to be rapidly generated for virtually any desired genomic locus by fluorescently labeling pooled amplicons after PCR with a flexibility and cost effectiveness that is otherwise unachievable with other methods (Supplementary Fig. 2a,b and Supplementary Note). As a proof of principle, we constructed a probe consisting of 50 200-nt unique fragments obtained by PCR and labeled with the Universal Linkage System7 targeting the HER2 (also known as ERBB2) locus on chromosome 17. The effective target size (ETS) of this probe (that is, the number of nucleotides effectively targeted) is 10 kb (50 amplicons × 200 nt), which is an order of magnitude shorter than the commercially available HER2 probes currently used in diagnostics. Hybridization with this HER2 probe was specific on both human lymphocyte metaphase spreads as well as in human mammary epithelial (HME) cells processed to preserve their three-dimensional nuclear structure a versatile genome-scale Pcr-based pipeline for high-definition dna fish