Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

Abstract

Successful PCR starts with proper priming between an oligonucleotide primer and the template DNA. However, the inevitable risk of mismatched priming cannot be avoided in the currently used primer system, even though considerable time and effort are devoted to primer design and optimization of reaction conditions. Here, we report a novel dual priming oligonucleotide (DPO) which contains two separate priming regions joined by a polydeoxyinosine linker. The linker assumes a bubble-like structure which itself is not involved in priming, but rather delineates the boundary between the two parts of the primer. This structure results in two primer segments with distinct annealing properties: a longer 5'-segment that initiates stable priming, and a short 3'-segment that determines target-specific extension. This DPO-based system is a fundamental tool for blocking extension of non-specifically primed templates, and thereby generates consistently high PCR specificity even under less than optimal PCR conditions. The strength and utility of the DPO system are demonstrated here using multiplex PCR and SNP genotyping PCR.

Figure 1.

Strategy and validation of DPO-based PCR. (A) Schematic representation of long conventional primer-based and DPO-based PCR strategies. (B) Comparison of 3′-RACE products of Ndufs2 obtained using long conventional primers (left) and DPO primers (right), containing different internal mismatched nucleotides at an annealing temperature of 60°C. Perfect match (lanes 1 and 4), 3 bp mismatches in the 3′-segment (lanes 2 and 5), and 3 bp mismatches in the 5′-segment (lanes 3 and 6). (C) Comparison of 3′-RACE products of Ndufs2 obtained using the perfectly matched long conventional primers (lanes 1 and 3) and DPO primers (lanes 2 and 4) at a low (55°C) and a high (65°C) annealing temperature. M, the 100-bp-size marker (Seegene). The primers are described in Table 1.

Figure 2.

Detection of multiple respiratory viruses. Comparison of PCR products generated using DPO-based multiplex RT-PCR (right) and long conventional primer-based multiplex RT-PCR (left) from five patient samples. N, negative control; P, positive control; Flu A, influenza A virus; Flu B, influenza B virus; RSV B, respiratory syncytial virus B; RSV A, respiratory syncytial virus A; OC43, coronavirus OC43. The primers are described in Table 1.

Figure 3.

Detection of an SNP in CYP2C19. Schematic representation of primer positions, and comparison of PCR products generated using DPO-based multiplex RT-PCR (right) and long conventional primer-based multiplex RT-PCR (left) from nine human blood samples. The asterisk indicates the position of the SNP in CY2C19. M, the 100-bp-size marker (Seegene). The primers are described in Table 1.