Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

Abstract

Real-time PCR assays have recently been developed for diagnostic and research purposes. Signal generation in real-time PCR is achieved with probe designs that usually depend on exonuclease activity of DNA polymerase (e.g. TaqMan probe) or oligonucleotide hybridization (e.g. molecular beacon). Probe design often needs to be specifically tailored either to tolerate or to differentiate between sequence variations. The conventional probe technologies offer limited flexibility to meet these diverse requirements. Here, we introduce a novel partially double-stranded linear DNA probe design. It consists of a hybridization probe 5'-labeled with a fluorophore and a shorter quencher oligo of complementary sequence 3'-labeled with a quencher. Fluorescent signal is generated when the hybridization probe preferentially binds to amplified targets during PCR. This novel class of probe can be thermodynamically modulated by adjusting (i) the length of hybridization probe, (ii) the length of quencher oligo, (iii) the molar ratio between the two strands and (iv) signal detection temperature. As a result, pre-amplification signal, signal gain and the extent of mismatch discrimination can be reliably controlled and optimized. The applicability of this design strategy was demonstrated in the Abbott RealTime HIV-1 assay.

Figure 1.

Partially double-stranded linear DNA probe design. (A) HP:QO duplex formation in the absence of targets leads to fluorescence quenching. HP consists of 20, 25, 31 or 43 bases while QO consists of 12, 14 or 16 bases. QO sequence is complementary to 5′ sequence of HP. (B) HP binding to target during PCR amplification restores fluorescent signal by replacing QO.

Figure 2.

Effects of HP length, QO length and QO concentration on HP:QO melting profile. FAM fluorescence intensity was measured at each temperature hold through the thermal cycling program, as described in Materials and Methods section. (A). Probes were formed with 81 nM HP31 alone (triangle) or together with 81 nM QO16 (square). (B). Probes were formed with 81 nM HP20 alone (triangle), or together with 81 nM (1:1) QO12 (circle), QO14 (diamond) or QO16 (square). (C). Probes were formed with 81 nM HP20 and QO16 at a concentration of 0 nM (1:0; triangle), 81 nM (1:1; square) or 243 nM (1:3; diamond). (D). Effect of QO length and HP:QO molar ratio on probe melting temperature (θ_m). HP:QO ratio was tested at either 1:1 (triangle) or 1:3 (square).

Figure 3.

Impact of QO on HP:Target melting profile (A and C) and on real-time RT-PCR (B and D). Melting profile: 81 nM HP20 (A) or HP31 (C) alone (open or closed triangle) or combined with 81 nM (1×) QO16 (open or closed square) was analyzed in melting experiment in the absence (open symbol) or presence (closed symbol) of 405 nM complement DNA. Real-time RT-PCR: 200 nM HP20 (B) or HP31 (D) alone (open square) or combined with 200 nM (1×) QO16 (open triangle) was used to detect target sequences in a real-time RT-PCR reaction. Three replicates of reactions were tested for each condition.

Figure 4.

Effects of HP length, QO length and concentration on mismatch discrimination/tolerance in melting and real-time RT-PCR. Melting profile: (A) HP20 alone; (C) HP20:QO16 (1:1); (E) HP20:QO16 (1:3); (G) HP20:QO12 (1:3); (I) HP31:QO16 (1:1). Melting experiment was performed for probe alone (open triangle), or in the presence of perfect-match targets (closed square), or targets containing a single mismatch at the 12th position (from 5′end of HP) (open square). Real-time RT-PCR: (B) HP20 alone; (D) HP20:QO16 (1:1); (F) HP20:QO16 (1:3); (H) HP20:QO12 (1:3); (J) HP31:QO16 (1:1). PCR amplification curves for perfect-match targets (open square) were compared with those for 1 mismatch targets (open triangle). The mismatch target had the same mismatch tested in the melting experiment. Three replicates of reactions were tested for each condition.

Figure 4.

Effects of HP length, QO length and concentration on mismatch discrimination/tolerance in melting and real-time RT-PCR. Melting profile: (A) HP20 alone; (C) HP20:QO16 (1:1); (E) HP20:QO16 (1:3); (G) HP20:QO12 (1:3); (I) HP31:QO16 (1:1). Melting experiment was performed for probe alone (open triangle), or in the presence of perfect-match targets (closed square), or targets containing a single mismatch at the 12th position (from 5′end of HP) (open square). Real-time RT-PCR: (B) HP20 alone; (D) HP20:QO16 (1:1); (F) HP20:QO16 (1:3); (H) HP20:QO12 (1:3); (J) HP31:QO16 (1:1). PCR amplification curves for perfect-match targets (open square) were compared with those for 1 mismatch targets (open triangle). The mismatch target had the same mismatch tested in the melting experiment. Three replicates of reactions were tested for each condition.

Figure 5.

Effects of read temperature on mismatch discrimination/tolerance in real-time RT-PCR. Impact of 1 mismatch on real-time RT-PCR was evaluated with HP20 at 35°C (A) and 56°C (B) and with HP31 at 35°C (C) and 56°C (D). The mismatch target was the same as used in Figure 4.

Figure 6.

Mismatch tolerance for HP43:QO14 (1:4) in melting analysis (A) and real-time PCR (B). Melting experiments were performed with HP43:QO14 (1:4) in the absence (open triangle) or presence of target with 0 (closed square), 1 (open square), 2 (open diamond), 3 (open circle) or 4 (closed triangle) mismatches in probe-binding region. Positions of mismatches are as follows: 1 mis is 12th or 27th nucleotide; 2 mis are the 9th and 12th, or 12th and 27th, or 3rd and 12th or 9th and 27th nucleotides; 3 mis are the 12th, 24th and 25th nucleotides; 4 mis are the 12th, 21st, 24th and 25th nucleotides. All mismatch nucleotide positions start from the 5′ end of HP43.