Coamplification at lower denaturation temperature-PCR increases mutation-detection selectivity of TaqMan-based real-time PCR.
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.
Li
Jin
J
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.
Wang
Lilin
L
Jänne
Pasi A
PA
Makrigiorgos
G Mike
GM
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
D015342:DNA Probes; D016159:Tumor Suppressor Protein p53