Parallel DNA polymerase chain reaction: Synthesis of two different PCR products from a DNA template

Abstract

Conventionally, in a polymerase chain reaction (PCR), oligonucleotide primers bind to the template DNA in an antiparallel complementary way and the template DNA is amplified as it is. Here we describe an approach in which the first primer binds in a parallel complementary orientation to the single-stranded DNA, leading to synthesis in a parallel direction. Further reactions happened in a conventional way leading to the synthesis of PCR product having polarity opposite to the template used. This is the first study showing that synthesis of DNA can happen also in a parallel direction. We report that from a single-stranded DNA template, two different but related PCR products can be synthesized.

Figure 1.. Schematic diagram showing PCR amplification of a single-stranded DNA by using conventional antiparallel oligonucleotide primers.

Figure 2.. Schematic diagram showing PCR amplification of a single-stranded DNA by using the PD-PCR (parallel DNA PCR) approach in which the first primer binds to the template DNA in a parallel complementary manner.

The second primer binds to the newly synthesized DNA in an antiparallel manner and later both primers amplify the new DNA in a conventional manner. PCR products obtained will have opposite polarity as compared to the template used.

Figure 3.. PD-PCR (parallel DNA PCR) and PCR: Lanes 1–6 show 120 bp PCR products amplified at annealing temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C, respectively, using conventional antiparallel complementary primers.

Lane 7 is 100 bp molecular weight marker and Lanes 8–13 show PCR products amplified by parallel DNA PCR scheme at annealing temperature of 45°C, 50°C, 55°C, 58°C, 60°C, 65°C, respectively. In all cases, denaturation was performed at 95°C for 15 seconds, annealing for 30 seconds while extension at 72°C for 30 second for a total of 30 cycles.

Figure 4.

( A): A control reaction showing that PCR products were obtained when both primers were added as per scheme in Figure 2. In Figure 4 (A), Lane 1 shows 120 bp PCR products synthesized by PD-PCR, while in Lanes 2 and 3, only single primers were added and as expected no PCR product was synthesized. Figure 4(B) shows a negative control reaction of conventional PCR and PD-PCR in which the template DNA was not added.

Figure 5.

DNA sequencing results. Sequencing results in ( A) show that 120 bp DNA was amplified as it is while sequencing results in ( B) confirm that PCR products were obtained as per the scheme shown in Figure 2.

Figure 6.

Real time PCR and PD-PCR ( A) show ampliflication plot and dissociation curves obtained after real time PCR analysis of amplification of 120 nucleotides single stranded template DNA via conventional PCR and PD-PCR. In control reaction no template DNA was added. ( B) PCR products obtained in real time PCR were also run on agarose gel and visualised on gel doc system.