During the analysis of the 5′ flanking region of the androgen receptor, we have used the PCR technique extensively to create mutations. The 5′ and 3′ end primers were designed to contain 16-17 nucleotides homologous to the template sequence, with restriction enzyme sites and flanking nucleotides upstream of the homologous sequences. Using these primers we have successfully created several nested deletions which were cloned into pBLCAT3 vectors and used in transient transfection analysis. On identification of a putative suppressor element we used the PCR megaprimer technique to delete a 28-bp sequence. The 5′ end oligonucleotide contained 18 nucleotides corresponding to sequences upstream of the putative suppressor element and another 18 nucleotides corresponding to sequences downstream of the putative suppressor element. The putative suppressor element was deleted in two successive PCR reactions.
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