The ability to detect low levels of sequence variants among mostly wild-type DNA is critical for early cancer detection, prenatal testing, and infectious diseases. In cancer, low-level mutations (<10%) are usually below the detection limits of most genotyping techniques, including standard sequencing. Many moderate- to high-sensitivity PCR-based methods have been developed over the past two decades to enrich minority alleles. Limiting the denaturation temperature of PCR can be used to enrich known and unknown mutations (1). However, careful control of cycling temperatures is necessary and multiple amplicons can seldom be analyzed on the same plate. Allele-specific PCR methods to enrich known mutations include the amplification refractory mutation system (2), mutant allele–specific amplification (3), and methods that incorporate locked nucleic acids for increased specificity (4,5). Alternatively, nucleic acid clamps can be used to block wild-type amplification, including peptide nucleic acid (6-9) and locked nucleic acid (10-12) probes. In some methods, primers that select mutant DNA are combined with probes that block wild-type amplification. In one version, the blocking probe competes with the allele-specific primer (13,14) for increased sensitivity. However, all these methods can result in false positives when sensitivities below 0.1% are attempted (13-15).

Herein we enhance allele-specific PCR with a competitive blocking probe by asymmetric amplification and probe melting analysis to increase sensitivity and specificity. Unlabeled probes (16,17), dual-hybridization probes (18,19), and molecular beacons (20) are used as probes and also identify rare alleles by melting analysis. The method is demonstrated on the BRAF point mutation p.V600E (c.1799 T > A), the most common change in papillary thyroid carcinoma (21) that occurs in more than 80% of cases studied (21,22).

Materials and methods

DNA samples

DNA from normal human blood and a homozygous BRAF c.1799T > A cell line (Cat. no. HTB-72; ATCC, Manassas, VA, USA) were extracted by salting out (Gentra, Puregene, Qiagen, Valencia, CA, USA). DNA concentrations and quality were assessed by absorbance (NanoDrop, Wilmington, DE, USA) with minor adjustments by PCR quantification cycle (Cq). Different mutation percentages (10%, 1%, 0.1%, 0.01%, 0.001%, and 0.0001%) were obtained by mixing.

Clinical samples were obtained from a total of 47 patients. Forty-four of these patients had samples with both surgical excision tissue and fine-needle aspirates of thyroid nodules while the remaining 3 patients had only needle aspirates. All samples were de-identified and blinded by ARUP Laboratories (Salt Lake City, UT, USA) after testing for BRAF c.1799A using dual-hybridization probes as described previously (23).

Oligonucleotides

The allele-specific primer 5′-GTGATTTTGGTCTAGCTACAGA-3′, reverse primer 5′-TCAGTGGAAAAATAGCCTCAATTC-3′ and unlabeled probe 5′-TCTAGCTACAGTGAAATCTCGATG-P-3′ were synthesized at the University of Utah core facility by standard phosphoramidite synthesis. Dual-hybridization probes 5′-AGCTACAGTGAAATCTCGATGGAG-fluorescein-3′ and 5′-LCRed640-GGTCCCATCAGTTTGAACAGTTGTCTGGA-P-3′ were synthesized by Idaho Technology (Salt Lake City, UT, USA). The molecular beacon 5′-FAM-CGGTCTAGCTACAGTGAAATCTCGACCG-BHQ1-3′ was synthesized by Biosearch Technologies, Novato, CA, USA). The underlined bases are matched to the c.1799T > A mutation, and “P” signifies a 3′-phosphate.

PCR and melting analysis

PCR was performed in 10-µL reactions containing 2 mmol/L MgCl₂, 50 mmol/L Tris (pH 8.3), 500 mg/L BSA, 200 µmol/L of each dNTP, 0.4 units KlenTaq polymerase (Ab Peptides, St. Louis, MO, USA), 64 ng/µL Anti-Taq monoclonal antibody (eENZYME, Montgomery Village, MD, USA), 0.5x LCGreen Plus (Idaho Technology), and 50 or 500 ng human genomic DNA. For symmetric PCR, both primers were at 0.5 µM. For asymmetric PCR, the allele-specific primer was reduced to 0.1 µM or 0.05 µM. In experiments with blocking probes, the allele-specific primer was at 0.05 µM with a probe concentration of 0.5 µM.

Eighty cycles of rapid-cycle PCR was performed on a LightCycler 1.5 (Roche Applied Science, Indianapolis, IN, USA) after an initial denaturation 95°C for 1 min, unless otherwise specified. When an unlabeled probe or no probes were used, each cycle consisted of a 95°C denaturation for 0 s and a 64°C annealing/extension phase for 4 s with programmed transitions of 20°C/s. Following PCR, a melting protocol from 55°C to 92°C with a 0.2°C/s ramp rate was used. The protocols for dual hybridization and molecular beacon probes were similar except the annealing/extension phase of PCR was 64°C for 0 s and a 0.1°C/s ramp between 50°C and 75°C was used for melting. The second derivative method (24) was used to determine quantification cycle (Cq) values (25).

Single-copy DNA amplification

Digital PCR was performed by diluting BRAF c.1799A genomic DNA to less than one copy per reaction in the presence or absence of wild-type DNA. Approximately 48 copies (0.144 ng) of c.1799A DNA were amplified across the 96 wells of the LC480 (Roche) after an initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 10 s and 64° for 15 s. In a separate experiment, ~10 copies (0.030 ng) of c.1799A DNA admixed with 3.2 x 106 copies (10 µg) wild-type genomic DNA (0.0003% mutant DNA) were amplified after separation into 20 reactions on a LightCycler 1.5, using the unlabeled probe cycling conditions.

Results and discussion

Allele-specific PCR is widely used to enrich rare alleles. Specificity is obtained by matching the 3′-end of one primer to the desired allele. Other alleles are mismatched and their amplifications are inhibited. Inhibition is not complete, however, and mismatched alleles are amplified to various extents depending on the type of mismatch (26,27). The lowest rare-allele percentage that can be distinguished from pure wild-type DNA establishes the sensitivity of the method and is conveniently studied by real-time PCR. Lowering the Mg^{2 +} and the primer concentrations can increase sensitivity and lower the false-positive rate (28).

We enhanced allele-specific PCR amplification and detection by using asymmetric PCR, a wild-type blocking probe, and probe melting analysis. Rare allele enrichment was optimal with an excess of blocking probe and reverse primer compared with the allele-specific primer. Using the BRAF c.1799A single base mutation, as the concentration of the allele-specific primer decreased from 0.5 µM to 0.1–0.05 µM (in the absence of blocking probes), specificity increased while PCR efficiency decreased. Increasing specificity was reflected during real-time PCR by the ΔCq between wild-type and BRAF DNA increasing from 10 to 19–20 cycles. Decreasing PCR efficiency was evidenced by an increase in the Cq of BRAF DNA by 12–15 cycles. Although PCR efficiency was affected by decreasing the concentration of either primer, only lower concentrations of the allele-specific primer increased specificity. In order to compensate for the lower PCR efficiency, 80 cycles were typically performed, but this required only 30 min using rapid-cycle PCR (29).

A lower concentration of allele-specific primer produces an excess of one product strand during asymmetric PCR. If melting probes complementary to the excess strand are included, melting signatures can be observed that are specific to the allele under the probe (16). Figure 1 displays derivative melting plots obtained after asymmetric PCR of pure wild-type or BRAF c.1799A DNA using an unlabeled wild-type blocker as the melting probe. The matched wild-type allele melts about 4°C higher than the mismatched mutant allele. Similar to allele-specific competitive blocker PCR (13), the wild-type probe preferentially binds to wild-type DNA and competes with primer binding. At the same time, the allele-specific primer is matched to and preferentially extends the rare allele. Both allele-specific extension and wild-type blocking enhance enrichment and sensitivity for detecting the rare allele.