page_title: P element
section_title: Inverse PCR
hierarchical_section_title: P element / Use in molecular biology / Analysis of mutagenesis products / Inverse PCR
caption_attribution_description: English: By Richard Wheeler (Zephyris) 2005; Analysis of DNA flanking a known insert by PCR (specifically with p elements).
context_page_description: P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.
  The P element encodes for the protein P transposase. Unlike laboratory strain females, wild type females are thought also to express an inhibitor to P transposase function, from the very same element. This inhibitor reduces the disruption to the genome caused by the P elements, allowing fertile progeny. Evidence for this comes from crosses of laboratory females with wild type males. In the absence of the inhibitor, the P elements can proliferate throughout the genome, disrupting many genes and killing progeny.
  P elements are commonly used as mutagenic agents in genetic experiments with Drosophila. One advantage of this approach is that the mutations are easy to locate. In hybrid dysgenesis, one strain of Drosophila mates with another strain of Drosophila producing hybrid offspring and causing chromosomal damage known to be dysgenic. Hybrid dysgenesis requires a contribution from both parents.
context_section_description: Isolate the fly genome.
  Undergo a light digest (using an enzyme [enzyme 1] known NOT to cut in the reporter gene), giving fragments of a few kilobases, a few with the insertion and its flanking DNA.
  Self ligate the digest (low DNA concentration to ensure self ligation) giving a selection of circular DNA fragments, a few with the insertion and its flanking DNA.
  Cut the plasmids at some point in the reporter gene (with an enzyme [enzyme 2] known to cut very rarely in genomic DNA, but is known to in the reporter gene).
  Using primers for the reporter gene sections, the DNA can be amplified for sequencing.
  The process of cutting, self ligation and re cutting allows the amplification of the flanking regions of DNA without knowing the sequence. The point at which the ligation occurred can be seen by identifying the cut site of [enzyme 1].
caption: Process of analysis of DNA flanking a known insert by PCR.
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