title: Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR.
abstract: BACKGROUND
  DNA genotyping with mutation-specific TaqMan(R) probes (Applied Biosystems) is broadly used in detection of single-nucleotide polymorphisms but is less so for somatic mutations because of its limited selectivity for low-level mutations. We recently described coamplification at lower denaturation temperature-PCR (COLD-PCR), a method that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences during the PCR. We demonstrate that combining COLD-PCR with TaqMan technology provides TaqMan genotyping with the selectivity needed to detect low-level somatic mutations.
  
  
  METHODS
  Minor-groove binder-based or common TaqMan probes were designed to contain a nucleotide that matches the desired mutation approximately in the middle of the probe. The critical denaturation temperature (T(c)) of each amplicon was then experimentally determined. COLD-PCR/TaqMan genotyping was performed in 2 steps: denaturation at the T(c), followed by annealing and extension at a single temperature (fast COLD-PCR). The threshold cycle was used to identify mutations on the basis of serial dilutions of mutant DNA into wild-type DNA and to identify TP53 (tumor protein p53) and EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] mutations in tumors.
  
  
  RESULTS
  COLD-PCR/TaqMan genotyping identified G>A mutations within TP53 exon 8 (codon 273 mutation hot spot) and C>T mutations within the EGFR gene (drug-resistance mutation T790M) with a selectivity improvement of 15- to 30-fold over regular PCR/TaqMan genotyping. A second round of COLD-PCR/TaqMan genotyping improved the selectivity by another 15- to 30-fold and enabled detection of 1 mutant in 2000 wild-type alleles. Use of COLD-PCR/TaqMan genotyping allowed quantitative identification of low-level TP53 and T790 mutations in colon tumor samples and in non-small-cell lung cancer cell lines treated with kinase inhibitors.
  
  
  CONCLUSIONS
  The major improvement in selectivity provided by COLD-PCR enables the popular TaqMan genotyping method to become a powerful tool for detecting low-level mutations in clinical samples.
authors.affiliation: Department of Radiation Oncology, Divisions of Genomic Stability and DNA Repair, and Medical Physics, Lowe Center for Thoracic Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Li
authors.forename: Lilin
authors.initials: L
authors.lastname: Wang
authors.forename: Pasi A
authors.initials: PA
authors.lastname: Jänne
authors.forename: G Mike
authors.initials: GM
authors.lastname: Makrigiorgos
mesh_terms: D045744:Cell Line, Tumor; D015342:DNA Probes / Q000032:analysis* / Q000235:genetics*; D005091:Exons / Q000235:genetics; D005838:Genotype; D006801:Humans; D008297:Male; D009154:Mutation / Q000235:genetics; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
chemical_list: D015342:DNA Probes; D016159:Tumor Suppressor Protein p53