Inverse PCR Background
Inverse PCR also called IPCR, and was first described by Ochman et al. in 1988 (1).
A limitation of standard PCR is that 5' and 3' flanking regions of your DNA fragment of interest must be known. Inverse PCR allows you to conduct PCR when you only have the information of one internal sequence.
Inverse polymerase chain reaction is a variant of PCR, and is used when only one internal sequence of the target DNA is known. It is therefore very useful in identifying flanking DNA sequences of genomic inserts. Similar to other PCR methods, inverse PCR amplifies target DNA using DNA polymerase.
Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usual orientation. The template for the reverse primers is a restriction fragment that has been ligated upon itself to form a circle.
The Inverse PCR Method
The inverse PCR method includes a series of digestions and self-ligations with the DNA being cut by a restriction endonuclease. This cut results in a known sequence at either end of unknown sequences.
Inverse PCR Steps
1) Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion.
2) Self-ligation is induced under low concentrations causing the phosphate backbone to reform. This gives a circular DNA ligation product.
3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within the known internal sequence generating a linear product with known terminal sequences. This can now be used for PCR (polymerase chain reaction).
4) Standard PCR is conducted with primers complementary to the now known internal sequences.
Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequences are digested and then ligated to generate circular DNA.
PCR primers pointing away from the known sequences are then employed to amplify the flanking sequences.
Applications of Inverse PCR
Inverse PCR has numerous applications in molecular biology including the amplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
Inverse PCR Protocol
Inverse PCR References