Inverse PCR uses standard PCR (polymerase chain reaction), however it has the primers oriented in the reverse direction of the usualorientation. 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 arestriction endonuclease. This cut results in a known sequence at either end of unknown sequences.
Inverse PCR Steps1) 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. Thisgives a circular DNA ligation product.3) Target DNA is then restriction digested with a known endonuclease. This generates a cut within theknown internal sequence generating a linear product with known terminal sequences. This can now beused for PCR (polymerase chain reaction).4) Standard PCR is conducted with primers complementary to the now known internal sequences.In summary: Inverse PCR functions to clone sequences flanking a known sequence. Flanking DNA sequencesare digested and then ligated to generate circular DNA. PCR primers pointing away from the knownsequences are then employed to amplify the flanking sequences.
Applications of Inverse PCR
Inverse PCR has numerous applications in molecular biology including theamplification and identification of sequences flanking transposable elements, and the identification of genomic inserts.
Reverse Transcription Polymerase Chain Reaction
Reverse transcription polymerase chain reaction (RT-PCR) is based on the polymerase chain reaction (PCR). More importantly it is based on the process of reverse transcription, which reverse transcribes RNAinto DNA and was initially isolated from retroviruses. The techniques of RT-PCR allows the formation of cDNA(complementary or copy DNA) from RNA, which stores the sequence of RNA (such as messenger RNA, mRNA) in the more stableform of nucleic acid, DNA. This reverse transcription from RNA into its reverse complement DNA (cDNA) is the first step of ausually two-step process of RT-PCR. Furthermore, by copying the RNA into DNA, one can then amplify the cDNA sequence byusing primers specific for the DNA sequence. This amplification is the final second major step of the two-step process of RT-PCR.