Experimental intra- and inter-assay variability of the basic real-time LCR protocol.

<p>The coefficient of variation (CV) was calculated from the measured mutant V₁₃₆ frequencies in the plasmid standards.</p

Schematic representation of the cycling steps for real-time LCR.

<p>LCR oligonucleotides are defined with colure purple for LCPR1, turquoise for LCPR2, green for LCPR3s and red for LCPR4s. PCR product used as LCR template is defined in deep blue.</p

Amplification plots of mutant DNA (V₁₃₆) detection in plasmid standard pools.

<p>(A) FAM fluorescent signals and (B) corresponding standard curve generated from plasmid standard pools with pre-defined mutant DNA concentrations. From left to right, curves represent 100%, (blue lines with circles), 25% (red lines with squares), 6.25% (green lines with triangles), 1.56% (grey lines with diamonds) and 0.39% (yellow lines with stars), V₁₃₆ frequencies (the 95% confidence limits for the standard curve are shown as hashed lines). Wild-type (A₁₃₆) samples are also included in the reaction and are placed in the far right (A) and are denoted by triangles in (B). Reactions were performed with 8 replicates. The threshold cycle (<i>Ct</i>) values (B) are plotted against the logarithm of the mutant DNA frequency (%).</p

Different real-time LCR protocols tested.

<p>Different thermocycling conditions, DNA ligases, length of oligonucleotides, gap modifications were examined. All trials were performed with the Mx3005P QPCR platform. Gap-A and gap-T LCR protocols used Platinum® <i>Taq</i> DNA polymerase.</p>a<p>Oligonucleotides used LCPR1, LCPR2, LCPR3s, LCPR4s.</p>b<p>Oligonucleotides used LCPR1, LCPR2, LCPR3L, LCPR4L.</p>c<p>Oligonucleotides used LCPR1, LCPR2G, LCPR3s, LCPR4s.</p>d<p>Oligonucleotides used LCPR1, LCPR2, LCPR3G, LCPR4s.</p>e<p>Optimal real-time LCR protocol.</p>f<p>The LOD and LOQ values are in % frequencies</p

Linear regression of the estimated mutant (V₁₃₆) log₁₀ frequencies on the actual log₁₀ frequencies.

<p>Four series of genomic DNA pools containing V₁₃₆ polymorphism at different frequencies (50%, 25%, 6.25%, 1.56%, and 0.39%) were tested by the basic real-time LCR protocol. Each sample was analysed in four replicates.</p

Oligonucleotides used in the different real-time LCR trials.

<p>The nucleotides which complement to the mutant SNP target are underlined on the discriminating oligonucleotides.</p>a<p>The melting temperature <i>T_m</i> was estimated using the DINAMelt web server (<a href="http://www.bioinfo.rpi.edu/applications/hybrid/hybrid2.php" target="_blank">http://www.bioinfo.rpi.edu/applications/hybrid/hybrid2.php</a>).</p>b<p>5′ phosphorylation on the ligating oligonucleotide.</p

Melting curve profile of the LCR products.

<p>The first peak represents the melting of oligonucleotide dimers and the second the melting of LCR products. Selected temperatures for LCR steps are indicated by arrows.</p

Representative results from gel electrophoresis analysis of genomic DNA from two different ovine blood samples extracted by eleven methods.

<p>Charge Switch gDNA Mini Tissue (lanes 1, 2), Nucleospin Blood (lanes 3, 4), Nucleospin Blood-Buffy Coat (lanes 5, 6), Modified Blood (lanes 7, 8), Nucleospin Tissue-Buffy Coat (lanes 9, 10), Modified Tissue (lanes 11, 12), Modified Dx (lanes 13, 14), Nucleospin Blood XL (lanes 15, 16), Phenol-Chloroform (lanes 17, 18), In-house (lanes 19, 20), Nucleospin Blood L (lanes 21, 22), M molecular weight marker l DNA/Hind III digest. </p

Representative results from gel electrophoresis analysis of genomic DNA from thirty different ovine blood samples extracted by four methods.

<p>Modified Blood (lanes 1 to 8), Modified Tissue (lanes 9 to 15), Modified Dx (lanes16 to 22), In-house (lanes 23 to 30), M molecular weight marker l DNA/Hind III digest. </p

Real-time PCR amplification plot of the gly-A gene, from <i>Campylobacter coli</i> spiked extracted samples from sheep and the controls containing only the <i>Campylobacter coli</i> DNA spike.

<p>Results from one sample with higher Ct value, extracted with the Phenol-Chloroform protocol is also shown indicating the presence of inhibitors. </p