Biotinylated probe isolation of targeted gene region improves detection of T790M epidermal growth factor receptor mutation via peptide nucleic acid-enriched real-time PCR.

The presence of the EGFR (epidermal growth factor receptor) T790M mutation in tumor tissue or body fluids from patients treated with EGFR tyrosine kinase inhibitors may indicate the onset of resistance to treatment. It is important to identify this mutation as early as possible so that treatment can be modified accordingly or potential side effects of further treatment can be avoided. This requirement calls for high detection sensitivity. Peptide nucleic acids (PNAs) are used as PCR clamps to inhibit amplification of wild-type DNA during PCR cycling, thereby enriching for rare mutations such as T790M. We describe a modification that improves the detection limit of PNA-clamp methods by at least 20-fold.We enriched the target by exposing genomic DNA to an EGFR exon 20-specific biotinylated oligonucleotide, followed by binding to streptavidin beads. We then prepared serial dilutions of the isolated target DNA containing the T790M mutation by mixing with wild-type DNA and then performed PNA clamp-based, real-time TaqMan PCR. For comparison, we performed PNA clamp-based PCR directly on genomic DNA.Whereas the detection limit for PNA clamp-based PCR performed directly on genomic DNA is 1 mutant allele in 1000 wild-type alleles, conducting the assay with biotinylated oligonucleotide-enriched target DNA improved the detection limit to 1 mutant allele in 40,000 wild-type alleles. A possible explanation for the improvement in detection is that biotin-based target isolation efficiently eliminates wild-type DNA; therefore, fewer erroneous amplifications of wild-type DNA can occur early during the PCR.Combining target molecule isolation via a biotinylated probe with PNA-enriched TaqMan real-time PCR provides a major improvement for detecting the EGFR T790M resistance mutation.