Fig 1.
Structure of a SuperSelective primer for the detection of BRAF V600E mutant sequences in the presence of abundant BRAF wild-type sequences, and a demonstration of its use in monoplex real-time PCR assays.
(A) SuperSelective primer BRAF V600E 24-14/14-5:1:1 contains a long 5'-anchor sequence that binds strongly to template strands, a short 3'-foot sequence that includes an interrogating nucleotide that is perfectly complementary to the corresponding nucleotide in a mutant template (but mismatches the corresponding nucleotide in a wild-type template), and a bridge sequence that links the anchor sequence to the foot sequence, and that is chosen to not be complementary to the corresponding intervening sequence in the template strand, thereby forming a single-stranded bubble that separates the function of the anchor from the function of the foot. (B) Real-time PCR assays employing SuperSelective primer BRAF V600E 24-14/14-5:1:1. Six reactions initiated with 106 BRAF wild-type templates plus different quantities of mutant templates (101, 102, 103, 104, 105, and 106) are plotted in blue; a reaction initiated with only 106 wild-type templates is plotted with a dotted orange line; and a control reaction containing no template DNA is plotted in red. (C) The threshold cycle measured for each reaction that contained mutant templates is plotted as a function of the logarithm of the number of mutant templates initially present in each reaction. The dotted orange line indicates the threshold cycle of the reaction containing only wild-type templates.
Fig 2.
Principle of operation of SuperSelective primers.
The selective step occurs only when a SuperSelective primer hybridizes to a DNA (-) template fragment present in the sample. Due to the small size of the foot sequence, the probability of initiation of a (+) amplicon is significantly greater if the target sequence of the foot in the (-) template fragment is a completely complementary mutant sequence, than if the target sequence of the foot in the (-) template fragment is a mismatched wild-type sequence. If (+) amplicon synthesis does occur, then the resulting (+) amplicon serves as a template for a conventional reverse primer, and is efficiently copied during the next thermal cycle, generating a (-) amplicon in which the complement of the unique bridge sequence that was present in SuperSelective primer is substituted for the intervening sequence that was present in the original (-) template fragment. As a result, in subsequent thermal cycles, the entire SuperSelective primer sequence is complementary to the (-) amplicon strands, and exponential amplification occurs efficiently, and can be followed in real-time.
Fig 3.
Primers utilized in monoplex PCR assays.
The bridge sequence within each SuperSelective primer is shown in blue, and the interrogating nucleotide in each foot sequence is shown in red. The EGFR L858R primers are arranged into groups that reflect their use in comparative experiments. The BRAF V600E target sequence is 69 base pairs long; the EGFR L858R target sequences are between 79 and 87 base pairs long, depending on the SuperSelective primer that is used; and the EGFR T790M target sequence is 68 base pairs long.
Fig 4.
SuperSelective primer elements that affect selectivity.
The linearity of the relationship between the threshold cycle and the logarithm of the initial number of mutant templates present in reactions containing 106 wild-type templates and different amounts of mutant template was determined for PCR assays initiated with different SuperSelective primer designs. (A) PCR reactions initiated with EGFR L858R SuperSelective primers possessing foot sequences of different length. (B) PCR reactions initiated with EGFR L858R SuperSelective primers that form symmetrical bubbles of different circumference.
Table 1.
Threshold cycles observed for PCR assays containing SuperSelective EGFR L858R primers whose interrogating nucleotide is located at different positions within the foot sequence.
Table 2.
Threshold cycles observed for PCR assays containing SuperSelective EGFR L858R primers that form bubbles with varying symmetries.
Fig 5.
Assays of samples containing human genomic DNA.
(A) To mimic assays initiated with DNA fragments isolated from blood plasma, we utilized SuperSelective primer EGFR L858R 24-14/14-5:1:1 in a set of eight PCR assays that were initiated with different quantities of digested human genomic DNA encoding the EGFR L858R mutation (DNA from 0; 10; 30; 100; 300; 1,000; 3,000; or 10,000 cells) in the presence of digested human genomic DNA from 10,000 wild-type human cells. The threshold cycle measured for each reaction that contained mutant templates is plotted as a function of the logarithm of the number of mutant templates initially present in each reaction. The dotted orange line indicates the threshold cycle of the reaction containing only wild-type templates. (B) Comparison of the number of EGFR T790M mutant target molecules present in human genomic DNA samples (as determined by droplet digital PCR) to the Ct values obtained for the same samples in real-time PCR assays that utilize SuperSelective primer EGFR T790M 24-14/14-4:1:1. The dotted orange line indicates the threshold cycle of a reaction containing only wild-type templates.
Fig 6.
Fine-tuned primers and molecular beacons utilized in multiplex PCR assays.
The BRAF SuperSelective primers whose sequences are shown here bind to the opposite strand to which the BRAF V600E SuperSelective primers shown in Fig 3 bind. The target sequence of the BRAF V600R SuperSelective primer mismatches the wild-type target sequence (and the BRAF V600E target sequence) at two adjacent nucleotides, so the BRAF V600R SuperSelective primer has two interrogating nucleotides. The BRAF target sequence is 136 base pairs long; and the EGFR target sequence is 90 base pairs long. The unique 32-nucleotide-long tag sequences at the 5' end of each SuperSelective primer is shown in green; the unique bridge sequence within each SuperSelective primer is shown in blue; and the interrogating nucleotides in the 3' foot sequences are shown in red. The complementary arm sequences of each molecular beacon are shown in black; and their probe sequences are shown in green. The differently colored fluorophores linked to the 5' ends of the molecular beacons are Quasar® 670, fluorescein, and CAL Fluor® Red 610; and the quencher moieties linked to the 3' ends of the molecular beacons are Black Hole Quencher®-1, and Black Hole Quencher®-2.
Fig 7.
Demonstration of the advantage of using non-symmetric primer concentrations (as opposed to using symmetric primer concentrations) in multiplex PCR assays that quantitate different mutations that, although occurring in different cells, are located in the same codon.
These assays contained fine-tuned SuperSelective primers BRAF V600E 32-30-10/9-6:1:1 and BRAF V600R 32-30-12/9-5:2:1 (where the underlined value reflects the length of the 5'-tag sequence), and two differently colored molecular beacon probes to detect the resulting amplicons. The left-hand panels show the results from the fluorescein-labeled BRAF V600R-specific molecular beacons (green curves); and the right-hand panels show the results from the Quasar® 670-labeled BRAF V600E-specific molecular beacons (blue curves). Samples without any templates were also run as controls (dotted orange curves). The symmetric PCR reactions, whose results are shown in the upper panels, contained 500 nM of each SuperSelective primer and 1,000 nM of the conventional BRAF common reverse primer. The non-symmetric PCR reactions, whose results are shown in the lower panels, contained only 60 nM of each SuperSelective primer and 1,000 nM of the conventional BRAF common reverse primer.
Fig 8.
Demonstration of the value of including primers for a reference wild-type gene in non-symmetric multiplex PCR assays employing fine-tuned SuperSelective primers.
The four left-hand panels show the results obtained for reactions containing 10,000 EGFR wild-type fragments, 10,000 BRAF wild-type fragments, 1,000 BRAF V600R fragments, and different quantities of BRAF V600E fragments (0; 10, 39; 156; 625; and 2,500 copies). The four right-hand panels show the results obtained for reactions containing 10,000 EGFR wild-type fragments, 10,000 BRAF wild-type fragments, 1,000 BRAF V600E fragments, and different quantities of BRAF V600R fragments (0; 10, 39; 156; 625; and 2,500 copies). The panels in the top row show the results recorded by the CAL Fluor® Red 610 EGFR wild-type-specific molecular beacon; the panels in the second row show the results recorded by the fluorescein-labeled BRAF V600R-specific molecular beacon; and the panels in the third row show the results recorded by the Quasar® 670-labeled BRAF V600E-specific molecular beacon. The lower panels plot the logarithm of the number of variable BRAF target fragments present in each reaction against the Ct value that was obtained. The Ct values for the 1,000 fragments of the non-variable BRAF mutant in each sample, as well as the Ct values for the 10,000 EGFR reference wild-type fragments present in each sample, are also plotted on the same line. The dotted orange lines indicate the Ct value of the sample containing no BRAF V600E mutant targets (left-hand panel) and no BRAF V600R targets (right-hand panel).
Table 3.
Comparison of Ct values obtained with SuperSelective EGFR L858R primers that can form mismatched 3'-terminal base pairs to the Ct values obtained with corresponding primers that cannot form 3'-terminal base pairs.