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 products ranging in size from < 1 to > 10 kb, regardless of target template sequence or structure, would facilitate several tasks in human genome research. Generation of a wide size range of PCR products would potentially expedite isolation of uncloned DNA (gaps) represented in physical maps and provide a means for maintaining order and orientation of closely linked loci during analysis. Long-range PCR offers an alternative to isolation of genomic or cDNA clones from tissues or species where appropriate libraries are unavailable. This methodology also provides a powerful strategy to pursue directed transposon-based mapping and sequencing of templates of 100 kb or greater. Further development of basic PCR technology has been necessary in order to make it feasible to use large DNA targets as primary templates for genome analysis. Application of these extended PCR capacities would potentially save time, materials, and cost.
Ohler
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LD
Advanced Center for Genetic Technology, Foster City, California 94404.
Zollo
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Mansfield
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Rose
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EA
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification*; D015226:Dinucleoside Phosphates / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005456:Fluorescent Dyes; D015723:Gene Library; D005798:Genes, Bacterial; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods*; D013698:Templates, Genetic; D014960:X Chromosome
D004247:DNA; D017931:DNA Primers; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D015226:Dinucleoside Phosphates; D005456:Fluorescent Dyes; C015772:CpG dinucleotide