The two principal determining steps in molecular diagnosis are the amplification and the identification steps. Accuracy of DNA amplification is primarily determined by the annealing sequence of the PCR primer to the analyte DNA. Accuracy for identification is determined either by the annealing region of a labelled probe for the real time PCR analysis, or the annealing of a sequencing primer for DNA sequencing analysis, that binds to the respective analyte (amplicon). Presently, housekeeping genes (Beta globin, GAPDH) are used in molecular diagnosis to verify that the PCR conditions are optimum, and are thus known as amplification controls [1], [2], [3], [4]. Although these genes have been useful as amplification controls, they lack the true definition of an internal control because the primers and annealing conditions are not identical to the analyte being assayed. This may result in a false negative report [5]. The IC-Code platform technology described here provides a true internal control where the internal control and analyte share identical PCR primers annealing sequences for the amplification step and identical sequencing primer annealing sequence for the identification step.The analyte and internal control have the same PCR and sequencing annealing sequences.This method assures for little or no false negatives and false positives due to the method’s design of using identical annealing conditions for the internal control and analyte, and by using DNA sequencing analysis for the identification step of the analyte, respectively.This method also allows for a set lower limit of detection to be used by varying the amount of internal control used in the assay.
Objective: This is an investigative study to evaluate a new companion diagnostic platform, allele specific multiplex sequencing (ASMS). Detection of Braf p.V600E from solid tumors is used as the test model with the following objectives: 1) whether ASMS can detect Braf p.V600E/K mutations from a variety of solid tumors, 2) whether ASMS can detect all Braf p.V600E from samples that were positive for Braf V600E by SNaPshot or Ion Torrent, and 3) whether ASMS can detect Braf p.V600E among samples that were reported negative by SNaPshot or Ion Torrent. Conclusions: ASMS assay detected all Braf p.V600E positives from different types of solid tumors that previously tested positive by SNaPshot or Ion Torrent. Further, ASMS was able to detect Braf p.V600E among samples that were reported negative by SNaPshot or Ion Torrent.
Methods: ASMS is a novel modification (US
We are presenting an evaluation of Allele Specific Multiplex Sequencing (ASMS) to detect two EGFR somatic mutations (L858R, T790M). Late stage lung cancer samples were tested for both EGFR mutations and were compared to either pyrosequencing or TruSeq. The analytical lower limit of detection (LLOD) for the ASMS-L858R assay was found to be 36 copies, and 72 copies for the ASMS-T790M assay. The forty-one FFPE samples that were tested for T790M showed 100% concordance with the respective comparative method. The forty-five FFPE samples tested previously by Truseq for L858R showed 100% concordance with ASMS. Out of the twenty L858R samples previously tested by pyrosequencing, there was 95% concordance with ASMS. Additionally, twenty-one normal blood samples were tested by ASMS were found to be negative for L858R and T790M. In conclusion, the detection of L858R and T790M by ASMS are in acceptable concordance with both pyrosequencing and TruSeq in detecting EGFR mutations from late stage lung cancer. Further, ASMS was able to detect EGFR (L858R) with 10 picograms (3 copies gDNA) of FFPE extracted DNA, and hence could be used to detect mutations from samples carrying low copy numbers.
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