Failure to amplify templates containing the synthetic loxP3 region using flanking primers.

2016-10-21T17:30:01Z (GMT) by Johan Kreuger Paul O’Callaghan
<p><b>A.</b> Primers targeting Wt sequences upstream (F<sup>Flank1</sup>) and downstream (R<sup>Flank1</sup>) of the synthetic loxP3 region were predicted to amplify a PCR product of 453 bp from the KO<sup>1st</sup> construct and 386 bp from Wt sequences. The 80 bp synthetic loxP3 region replaces eleven Wt-specific bps; therefore, this represents a unique identifier for amplicons derived from Wt alleles. <b>B.</b> PCR with these primers is predicted to produce 453 bp and 386 bp products from KO<sup>1st</sup> chimeras and a single 386 bp product from Wt samples. <b>C.</b> A single product is amplified from KO<sup>1st</sup> chimeras and Wt mice samples using the F<sup>Flank1</sup> with R<sup>Flank1</sup> primers. PCR was performed with an annealing temperature of 56°C and products were separated on a 1% agarose gel containing GelRed stain. <b>D.</b> Sanger sequencing of the PCR products from C confirms that only amplicons containing the Wt-specific identifier were produced. <b>E.</b> PCR with F<sup>loxP3</sup> and R<sup>Flank1</sup> primers using DNA isolated from four F1 progeny (derived from crossing male and female KO<sup>1st</sup> chimeras) identified two individuals as positive for the KO<sup>1st</sup> template. <b>F</b>. PCR with F<sup>Flank1</sup> with R<sup>Flank1</sup> primers failed to distinguish the KO<sup>1st</sup> positive F1 progeny from Wt samples. PCR was performed with an annealing temperature of 60°C and products were separated on a 2.5% agarose gel containing GelRed stain. The two KO<sup>1st</sup> lanes in C represent PCR products derived from the same two distinct KO<sup>1st</sup> chimeric mice presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165012#pone.0165012.g001" target="_blank">Fig 1</a>. The Wt and KO<sup>1st</sup> chimera DNA samples in F are isolated from brain tissue; these samples are also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0165012#pone.0165012.g003" target="_blank">Fig 3</a>. <i>Abbreviations</i>: KO<sup>1st</sup>: Knockout first chimera, F1: Filial hybrid 1, P: Primers only.</p>