Real-Time Bidirectional Pyrophosphorolysis-Activated Polymerization for Quantitative Detection of Somatic Mutations
نویسندگان
چکیده
Detection of somatic mutations for targeted therapy is increasingly used in clinical settings. However, due to the difficulties of detecting rare mutations in excess of wild-type DNA, current methods often lack high sensitivity, require multiple procedural steps, or fail to be quantitative. We developed real-time bidirectional pyrophosphorolysis-activated polymerization (real-time Bi-PAP) that allows quantitative detection of somatic mutations. We applied the method to quantify seven mutations at codons 12 and 13 in KRAS, and 2 mutations (L858R, and T790M) in EGFR in clinical samples. The real-time Bi-PAP could detect 0.01% mutation in the presence of 100 ng template DNA. Of the 34 samples from the colon cancer patients, real-time Bi-PAP detected 14 KRAS mutant samples whereas the traditional real-time allele-specific PCR missed two samples with mutation abundance <1% and DNA sequencing missed nine samples with mutation abundance <10%. The detection results of the two EGFR mutations in 45 non-small cell lung cancer samples further supported the applicability of the real-time Bi-PAP. The real-time Bi-PAP also proved to be more efficient than the real-time allele-specific PCR in the detection of templates prepared from formalin-fixed paraffin-embedded samples. Thus, real-time Bi-PAP can be used for rapid and accurate quantification of somatic mutations. This flexible approach could be widely used for somatic mutation detection in clinical settings.
منابع مشابه
Detection of extremely rare alleles by bidirectional pyrophosphorolysis-activated polymerization allele-specific amplification (Bi-PAP-A): measurement of mutation load in mammalian tissues.
Pyrophosphorolysis-activated polymerization (PAP) was developed to detect extremely rare mutations in complex genomes. In theory, PAP can detect a copy of a single base mutation present in 3 x 10(11) copies of the wild-type allele. In practice, the selectivity of detection is limited by a bypass reaction involving a polymerase extension error from the unblocked oligonucleotide annealed to the o...
متن کاملPyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma.
PURPOSE To develop a molecular tool to detect circulating tumor-derived DNA (ctDNA) in the plasma from patients with uveal melanoma as a marker of tumor burden and monitor treatment efficacy. EXPERIMENTAL DESIGN A real-time PCR was developed on the basis of bidirectional pyrophosphorolysis-activated polymerization (bi-PAP) for the quantification of ctDNA using 3'blocked primer pairs specific ...
متن کاملPyrophosphorolysis-activatable oligonucleotides may facilitate detection of rare alleles, mutation scanning and analysis of chromatin structures.
Pyrophosphorolysis-activated polymerization (PAP) was initially developed to enhance the specificity of allele-specific PCR for detection of known mutations in the presence of a great excess of wild-type allele. The high specificity of PAP derives from the serial coupling of pyrophosphorolysis-mediated activation of a pyrophosphorolysis-activatable oligonucleotide (P*) followed by extension of ...
متن کاملMultiplex dosage pyrophosphorolysis-activated polymerization: application to the detection of heterozygous deletions.
Large heterozygous chromosomal deletions and gene duplications are important classes of mutations that are generally missed by standard PCR amplification and sequencing. Multiplex dosage pyrophosphorolysis-activated polymerization (MD-PAP), a derivative of PAP, was utilized to detect these types of mutations. PAP is a method for nucleic acid amplification in which 3' blocked oligonucleotides (P...
متن کاملPyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification.
To measure mutation load or to detect minimal residual disease, a robust method for identifying one mutant allele in the range of 10(6)-10(9) wild-type alleles would be advantageous. Herein, we present evidence that pyrophosphorolysis-activated polymerization (PAP) has the potential to provide a highly specific and robust method of allele-specific amplification if DNA polymerases with higher py...
متن کامل