Pro12Ala polymorphism of the peroxisome proliferator-activated receptor γ2 in patients with fatty liver diseases

Figure 1 Cloning strategy and validation of a reference system. A: Cloning strategy for the generation of a reference system. The Pro12Ala mutation locus of the peroxisome proliferator-activated receptor-γ gene was synthesized as the proline or the alanine encoding oligonucleotides sequence. The proline or the alanine codon is indicated in yellow. The chemically synthesized oligonucleotide dimers, flanked by the overhangs of the EcoRI and the SpeI restriction sites (red/green), respectively, were used for insertion into pBluescript SKII plasmids; B: Real-time polymerase chain reaction (PCR) of the Pro12Ala locus depending on different copy numbers of reference sequences. Real-time PCR was performed using the LNA probes (Table 2) specific for the proline encoding sequence or the alanine encoding allele, respectively. 10⁶, 10⁵, 10⁴, 10³, 10², 10 copies of the plasmid reference sequences encoding either the Ala12 or the Pro12 locus were each diluted in 10 ng salmon sperm DNA and used for LNA probe based real-time PCR assays. Up to 10 copies per ng total DNA of both reference sequences were efficiently detected by the corresponding LNA probe labelled either by Hex or Fam fluorochrome.