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

John Skiadas, Christopher Aston, Akhtar Samad, Thomas S. Anantharaman, Bud Mishra, David C. Schwartz

Research output: Contribution to journalArticle

Abstract

Optical mapping is an approach for the rapid, automated, non- electrophoretic 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 mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps 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 Seal, XmnI, HpaI, ClaI, or BgfII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genOmiC loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.

Original languageEnglish (US)
Pages (from-to)1005-1009
Number of pages5
JournalMammalian Genome
Volume10
Issue number10
DOIs
StatePublished - 1999

ASJC Scopus subject areas

  • Genetics

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    Skiadas, J., Aston, C., Samad, A., Anantharaman, T. S., Mishra, B., & Schwartz, D. C. (1999). Optical PCR: Genomic analysis by long-range PCR and optical mapping. Mammalian Genome, 10(10), 1005-1009. https://doi.org/10.1007/s003359901148