NOVEL PCR TECHNIQUE USING ALU-SPECIFIC PRIMERS TO IDENTIFY UNKNOWN FLANKING SEQUENCES FROM THE HUMAN GENOME

The rapid and reproducible identification of new cellular DNA sequence s adjacent to known sequences is difficult to achieve with the current ly available procedures. Here we describe a novel approach based on th e polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesir able amplifications between Alu sequences, primers are constructed wit h dUTPs and destroyed by uracil DNA glycosylase treatment after 10 ini tial cycles of amplification. Only desirable fragments are then furthe r amplified with specific primers to the known region and to a tag seq uence introduced in the Alu-specific primer. Using this protocol, we h ave successfully identified cellular sequences flanking integrated hep atitis B virus DNA from the human genome of three hepatoma tissues. Th e method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based proto cols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicabl e to other studies of the human genome. (C) 1995 Academic Press, Inc.