Figure 1.
The principle of ligation detection reaction.
The discriminating probe (DP) and the common probe (CP) are designed to hybridize adjacently on the template DNA and are joined by ligase in the presence of a matching template (HPV PCR product). The discriminating 3′ base can be A, C, T or G. The reaction is thermally cycled and ligation products addressed on microarray spots by the unique ZipCode sequences flanking each CP. Hybridization control probe carries a different label (6-Fam) than the DP (Cy3).
Figure 2.
Specificity of HPV LDR probe pool.
The HPV LDR probe pool was tested against individual plasmid templates. The horizontal axis shows the probes by ZipCodes and corresponding HPV types. The vertical axis shows plasmid templates by HPV type. The signals are medians over spot replicates on 1–3 microarrays. The majority of the probes give a high hybridization signal only for their specific target plasmids. For HPV 11, 42 and 43, there are no functional probes. HPVs 39, 56 and 68 have only one functional probe each. The probe A85 (HPV 73 ) shows a strong nonspecific hybridization signal to HPV 44 template. Minor nonspecific hybridisation signals are evident in probes A59, A110, A57 and A56.
Figure 3.
Comparison of the PGMY-t and the original PGMY multiplex PCR primer mixes as measured by HPV LDR signals on microarray.
Both primer mixes at 0.2 µM primer concentration amplify all five HPV types from 1 ng of template (A) & (B) and from 1 pg of template (C) & (D). With the original PGMY mix at 1 ng template concentration, HPV 59 gives a false positive signal (B). HPV 33 signal is also relatively weak with the original PGMY at 1 pg template concentrations (D). Data are presented as means±SD from two independent microarrays. Y-axis shows signal intensity in arbitrary units. Asterisk (*) indicates the target HPV types present in the experiment.
Figure 4.
Evaluation of positive and negative microarray signals.
A) Distributions of background signals and signals from true positive probes (green) and true negative probes (red). For clarity, frequencies above 1000 and tails of intensity distributions over 2000 are not shown. B) P-value distributions of three statistical tests. Positive (pos) and negative (neg) probes are tested against the background distribution.
Table 1.
Analysis of patient samples with LDR and Hybrid Capture 2.
Table 2.
Analysis of patient samples with Roche Linear Array, LDR and Qiagen Hybrid Capture 2.