Sanger & qPCR

Base-Seq HDx™ Reference Standards

Overview

DNA sequencing data generated using the Sanger method is often limited by poor quality in the first 15-40 bases of the sequence due to primer binding and by a maximum possible read length of about 700-900 bases with significant deterioration in quality thereafter.

Further to this, use of Sanger sequencing to detect nucleotide polymorphisms requires that secondary alleles are present in at least 20% frequency, leaving a potential for false negative results in samples around or below this frequency.

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Case Study

EntroGen use Horizon Reference Standards to understand the limit of detection of their PCR based mutation analysis assays.

The work shows how Horizon FFPE reference materials are appropriate for determining the analytical sensitivity and specificity of mutation screening assays, and highlights the value of relevant reference samples for standardizing molecular assays.

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Variability In Assay Results

There is the potential therefore for errors to creep into assay results. This has been highlighted in a recent worldwide EGFR proficiency testing scheme (EQA) 2014, in which over 20% of the participating laboratories failed 1.

Figure 1 - S.Patton et al. Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing.

In the laboratory it is often assumed that a failed assay is as a result of a failed analytical step, there are multiple sources of variability in any molecular assay workflow and frequently the root cause is a problem with DNA extraction or issues with DNA quantification, leading to the assay being carried out with an insufficient amount of DNA.

To identify and control this variability, Horizon Discovery has developed two formats of rReference standards: Formalin-Fixed, Paraffin-Embedded (FFPE) sections for your workflow and Genomic DNA (gDNA) for your assay.

  • Precisely engineered cell lines containing mutations at the endogenous loci
  • Fully representative of a patient tumor
  • Establish your absolute limit of detection for every mutation
  • Determine the specificity and sensitivity of your assay with ease
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