An improved PCR method for walking in uncloned genomic DNA is described. Traditional methods like inverse PCR, randomly primed PCR, and adaptor ligation PCR have been used for walking in cloned DNA but are inefficient for uncloned DNA. The authors developed a method combining 'vectorette PCR' with 'suppression PCR' to enhance efficiency and accuracy. The method uses a special adaptor with a blunt end and an amine group on the 3'-end of the lower strand, which blocks polymerase extension and prevents primer binding unless a gene-specific primer extends a DNA strand opposite the upper strand of the adaptor. This results in the formation of 'panhandle' structures that suppress non-specific amplification. The adaptor is ligated to DNA fragments generated by restriction enzymes, and PCR is performed using adaptor primers and gene-specific primers. The method allows for the amplification of long DNA fragments with high specificity. The authors tested the method by walking upstream from exon 1 of the human tissue-type plasminogen activator (TPA) gene and obtained single major PCR products of known sizes. The results were verified by restriction enzyme digestion, confirming the gene-specificity of the walk. The method was also applied to the human transferrin receptor gene, yielding clean results. The improved method is valuable for rapidly finding promoters and regulatory elements, determining exon-intron boundaries, and walking upstream or downstream from sequence tagged sites. The method's high specificity is attributed to the suppression effect of the adaptor/adaptor primer combination.An improved PCR method for walking in uncloned genomic DNA is described. Traditional methods like inverse PCR, randomly primed PCR, and adaptor ligation PCR have been used for walking in cloned DNA but are inefficient for uncloned DNA. The authors developed a method combining 'vectorette PCR' with 'suppression PCR' to enhance efficiency and accuracy. The method uses a special adaptor with a blunt end and an amine group on the 3'-end of the lower strand, which blocks polymerase extension and prevents primer binding unless a gene-specific primer extends a DNA strand opposite the upper strand of the adaptor. This results in the formation of 'panhandle' structures that suppress non-specific amplification. The adaptor is ligated to DNA fragments generated by restriction enzymes, and PCR is performed using adaptor primers and gene-specific primers. The method allows for the amplification of long DNA fragments with high specificity. The authors tested the method by walking upstream from exon 1 of the human tissue-type plasminogen activator (TPA) gene and obtained single major PCR products of known sizes. The results were verified by restriction enzyme digestion, confirming the gene-specificity of the walk. The method was also applied to the human transferrin receptor gene, yielding clean results. The improved method is valuable for rapidly finding promoters and regulatory elements, determining exon-intron boundaries, and walking upstream or downstream from sequence tagged sites. The method's high specificity is attributed to the suppression effect of the adaptor/adaptor primer combination.