Inverse Polymerase Chain Reaction

Abstract

We describe a procedure that extends the utility of the polymerase chain reaction (PCR) in allowing the geometric amplification of an unknown DNA sequence that flanks a core region of known sequence. DNA containing the core region is digested with appropriate restriction enzymes to produce a fragment of suitable size for PCR amplification. The ends of the fragment are then ligated to form a circular molecule. Primers for PCR are homologous to the ends of the core region included within the circle, but oriented such that chain elongation proceeds across the uncharacterized region of the circle rather than across the core region separating the primers. This “inverse PCR” procedure can be used to amplify the sequences that originally flanked the core sequence. Inverse PCR has applications in producing probes of anomymous sequences or in determining the sequences of upstream and downstream flanking regions themselves.