Inverse polymerase chain reaction

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Inverse polymerase chain reaction is a variant of polymerase chain reaction (PCR) when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. Like other polymerase chain reaction processes, inverse polymerase chain reaction is used to amplify DNA samples, via the temperature-mediated enzyme DNA polymerase.

However, one of the key limitations of conventional polymerase chain reaction is that it requires primers complementary to the termini of the target DNA. This method allows PCR when only one internal sequence is known.

Inverse PCR involves a series of digestions and self-ligation before cutting by an endonuclease, resulting in known sequences at either end of the unknown sequence.

1. The target DNA is lightly digested into fragments of a few kilobases by an endonuclease.
2. Under low DNA concentration, self-ligation is induced, reforming the phosphate backbone, and giving a circular DNA product.
3. The target DNA is digested by an endonuclease known to cut once within the known internal sequence. This gives a linear product with known terminal sequences, suitable for polymerase chain reaction.
4. Polymerase chain reaction is carried out as usual, with primers complementary for sections of the known internal sequence.