Optical PCR: genomic analysis by long-range PCR and optical mapping.
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 ScaI, XmnI, HpaI, ClaI, or BglII. 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.
Skiadas
J
J
W.M. Keck Laboratory for Biomolecular Imaging, New York University, Department of Chemistry, Room 866, 31 Washington Place, New York, New York 10003, USA.
Aston
C
C
Samad
A
A
Anantharaman
T S
TS
Mishra
B
B
Schwartz
D C
DC
D004262:DNA Restriction Enzymes / Q000378:metabolism; D015894:Genome, Human; D006801:Humans; D007091:Image Processing, Computer-Assisted; D055095:Optics and Photonics; D020161:Physical Chromosome Mapping / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D015183:Restriction Mapping / Q000379:methods*; D010959:Tissue Plasminogen Activator / Q000235:genetics
D004262:DNA Restriction Enzymes; D010959:Tissue Plasminogen Activator