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Use of a simplified single-site PCR to facilitate cloning of genomic DNA sequences flanking a transgene integration site.

Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.

Abstract

We have used a simplified single-site PCR based strategy to isolate genomic DNA flanking the transgenic insert in a line of transgenic mice. The flanking sequences, which were refractory to more conventional cloning techniques, were isolated and characterized within 1 week. This strategy begins with the annealing and ligation of a single-stranded oligonucleotide to recessed 5' ends of restriction endonu-clease-digested, size-selected transgenic DNA. Following denaturation, a second oligonucleotide is used to prime DNA synthesis from a position within the transgenic sequences to the end of the genomic restriction fragment, finally synthesizing a complement of the ligated oligonucleotide sequence. Subsequently, a PCR is initiated which uses primers specific for the transgenic DNA and the newly synthesized DNA complementary to the ligated oligonucleotide. The only prerequisite data are sequence information for the transgenic DNA and Southern information regarding the size(s) of restriction fragments that contain the flanking genomic material. This report demonstrates one utility of this technique--enabling rapid isolation of specific mammalian genomic DNA sequences.

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