The authors present an improved PCR method for walking in uncloned genomic DNA, which is a challenging task due to the complexity and inefficiency of existing methods. They evaluate two initial approaches, "unpredictably primed PCR" and "vectorette PCR," but find them insufficient due to the generation of multiple PCR products. To address this, they develop a new method combining "vectorette PCR" with "suppression PCR." This method involves ligating a special adaptor to DNA fragments generated by specific restriction enzymes, followed by PCR using adaptor and gene-specific primers. The suppression PCR technology prevents non-specific amplification by forming 'panhandle' structures, ensuring high specificity. The method is successfully applied to walking upstream from exon I of the human tissue-type plasminogen activator (TPA) gene, generating single major PCR products of various sizes. The authors also demonstrate its utility in walking upstream from 5'-end coding regions of the human transferrin receptor gene. This improved method is valuable for identifying promoters, regulatory elements, and exon-intron boundaries in uncloned genomic DNA.The authors present an improved PCR method for walking in uncloned genomic DNA, which is a challenging task due to the complexity and inefficiency of existing methods. They evaluate two initial approaches, "unpredictably primed PCR" and "vectorette PCR," but find them insufficient due to the generation of multiple PCR products. To address this, they develop a new method combining "vectorette PCR" with "suppression PCR." This method involves ligating a special adaptor to DNA fragments generated by specific restriction enzymes, followed by PCR using adaptor and gene-specific primers. The suppression PCR technology prevents non-specific amplification by forming 'panhandle' structures, ensuring high specificity. The method is successfully applied to walking upstream from exon I of the human tissue-type plasminogen activator (TPA) gene, generating single major PCR products of various sizes. The authors also demonstrate its utility in walking upstream from 5'-end coding regions of the human transferrin receptor gene. This improved method is valuable for identifying promoters, regulatory elements, and exon-intron boundaries in uncloned genomic DNA.