USE OF A SENSITIVE FLUORESCENT INTERCALATING DYE TO DETECT PCR PRODUCTS OF LOW COPY NUMBER AND HIGH-MOLECULAR-WEIGHT

The ability to routinely and specifically amplify and detect PCR produ cts ranging in size from <1 to >10 kb, regardless of target template s equence or structure, would facilitate several tasks in human genome r esearch.(1,2) Generation of a wide size range of PCR products would po tentially expedite isolation of uncloned DNA (gaps) represented in phy sical maps and provide a means for maintaining order and orientation o f closely linked loci during analysis. Long-range PCR offers an altern ative to isolation of genomic or cDNA clones from tissues or species w here appropriate libraries are unavailable. This methodology also prov ides a powerful strategy to pursue directed transposon-based mapping a nd sequencing of templates of 100 kb or greater.(1-4) Further developm ent of basic PCR technology has been necessary in order to make it fea sible to use large DNA targets as primary templates for genome analysi s. Application of these extended PCR capacities would potentially save time, materials, and cost.