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As low as 0.86 fM mutant DNA was detected by this assay, and positive mutation detection was achieved with a wild-type to mutant ratio of 10,000 1.
In this study, an rolling circle amplification (RCA) based electrochemiluminescence (ECL) assay for highly sensitive point mutation detection was developed.
Mutation detection was performed by melting peak analysis of the probe PCR product hybrid performed on a LightCycler (Roche Diagnostic).
Then a single-stranded DNA (ssDNA) probe for BRCA1 5382 insC mutation detection was immobilized on the modified electrode for a specific time.
In validation studies using an analytical mutation standard, the sensitivity of the assay for EGFR mutation detection was at least 0.1% and specificity was 100%.
In addition, the positive mutation detection was achieved with a wild-type to mutant ratio of 10 000:1, due to the high fidelity of DNA ligase in differentiating mismatched bases at the ligation site.
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The positive mutation detection is achieved with a wild-type to mutant ratio of 5000 1.
Mutation detection is complex, due to the large gene size, the large variety of mutations and the presence of pseudogenes.
Multiple sequence alignments and mutation detection were performed as previously described [38], [52].
In practice, patients with tumors in which the analysis failed will receive anti-EGFR therapy, suggesting that efficient mutation detection is important.
KRAS mutation detection is summarized in Table 1.
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