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The polymerase dynamics of each was analyzed by SMRT sequencing and compared with a control template of the same sequence but lacking the modifications.
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Primer pairs efficiency values were determined from amplifications of a series of dilutions of a control template in which all possible BamHI/BglII ligation products of the PADI gene locus should be equally represented, generated from 30 µg of the BAC preparation, restricted and ligated at a high concentration (300 ng/µl).
We demonstrated that our primers perform as good as hand-crafted primers on a control template from random ligations of yeast genomic DNA.
We had previously demonstrated that a partially purified fraction containing all three coactivators (NT2 P1M, see Figure 1 figure supplement 1) is required for OCT4/SOX2 activated transcription of Nanog but has no effect on basal or Sp1-activated transcription of a control template in vitro (Fong et al., 2011).
To create a pool of all possible interacting products, control templates of defined molar amounts were prepared.
qRT-PCR analysis of cDNAs for each gene included a negative control template generated without addition of reverse transcriptase during cDNA synthesis.
Quantification was achieved using serial dilutions of a 3C-positive control template on each plate.
Quantitative values of the telomerase activity were obtained from a standard curve generated by using dilutions of control template provided by the kit.
Each run included a control free of template to test the assay reagents for contamination.
To this end, the paper first describes generic vertical and horizontal management control templates: archetypes of management control.
The sensitivity and specificity of these assays was higher than individual assays based on direct antigen detection and standard PCR against a range of control templates and in several hundred clinical specimens.
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