A RELIABLE AMPLIFICATION TECHNIQUE WITH SINGLE-SIDED SPECIFICITY FOR THE ISOLATION OF 5' GENE-REGULATING REGIONS

A simple and efficient method is described for the isolation of extens ion fragments of known DNA sequences by polymerase chain reaction (PCR ) using a single specific primer. With this method, size-selected geno mic DNA fragments are ligated to a plasmid Vector (pGEM-4Z) which cont ains sequencing primers and the population of chimeric plasmids is use d for transforming Escherichia coli. DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-speci fic primer and either of the standard sequencing primers of the plasmi d vector. This method appears to be more Versatile than inverse PCR (I PCR), since: ii) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be u sed in PCR are available in high amount, thus facilitating all manipul ations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites c an be chosen from the vector polylinker. Using this method, we have is olated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt seq uence of the 5' region of the dhfr-ts cDNA clone as the specific prime r.