Inverse Polymerase Chain Reaction (PCR).

Abstract

The standard polymerase chain reaction (PCR) is used to amplify a segment of DNA that lies between two inward-pointing primers. In contrast, inverse PCR (also known as inverted or inside-out PCR) is used to amplify DNA sequences that flank one end of a known DNA sequence and for which no primers are available. Inverse PCR DNA involves digestion by a restriction enzyme of a preparation of DNA containing the known sequence and its flanking region. The individual restriction fragments (many thousands in the case of total mammalian genomic DNA) are converted into circles by intramolecular ligation, and the circularized DNA is then used as a template in PCR. The unknown sequence is amplified by two primers that bind specifically to the known sequence and point in opposite directions. The product of the amplification reaction is a linear DNA fragment containing a single site for the restriction enzyme originally used to digest the DNA. This site marks the junction between the previously cloned sequence and the flanking sequences. The size of the amplified fragment depends on the distribution of restriction sites within known and flanking DNA sequences.