Optical PCR: Genomic analysis by long-range PCR and optical mapping

Optical mapping is an approach for the rapid, automated, non-electrophoreti c construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical map ping and long-range polymerase chain reaction (PCR), in a process we term o ptical PCR, which enables automated construction of ordered restriction map s of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high -resolution maps was generated, which agreed closely with maps predicted fr om sequence data. The data demonstrate an approach to construct physical ma ps of genomic loci where very little prior sequence information exists, sin ce the only sequence needed is that required to anchor PCR primers. Large s egments of genomic DNA (within the practical limits imposed by long-range P CR) can be mapped quickly and to high resolution without the use of cloning vectors.