Skip to main page content (VSports手机版)
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely. V体育官网.

. 2007;35(6):e40.
doi: 10.1093/nar/gkm051. Epub 2007 Feb 7.

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

Jong-Yoon Chun  1 , Kyoung-Joong Kim, In-Taek Hwang, Yun-Jee Kim, Dae-Hoon Lee, In-Kyoung Lee, Jong-Kee Kim
Affiliations

"V体育ios版" Dual priming oligonucleotide system for the multiplex detection of respiratory viruses and SNP genotyping of CYP2C19 gene

"V体育官网" Jong-Yoon Chun et al. Nucleic Acids Res. 2007.

"V体育平台登录" 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 VSports手机版. 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. .

PubMed Disclaimer

Figures

Figure 1.
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.
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.
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.
See this image and copyright information in PMC

References

    1. Birch DE, Kolmodin J, Wong J, Zangenberg GA, Zoccoli MA, McKinney N, Young KKK, Laird WJ. The use of a thermally activated DNA polymerase PCR gives improved specificity, sensitivity and product yield without additives or extra process steps. Nature. 1996;381:445–446. - PubMed
    1. Brownie J, Shawcross S, Theaker J, Whitcombe D, Ferrie R, Newton C, Little S. The elimination of primer-dimer accumulation in PCR. Nucleic Acids Res. 1997;25:3235–3241. - PMC (V体育ios版) - PubMed
    1. Ailenberg M, Silverman M. Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS) Biotechniques. 2000;29:1018–1020. 1022–1024. - PubMed
    1. Bergstrom DE, Zhang P, Johnson WT. Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5′- d(CGCXAATTYGCG)-3′. Nucleic Acids Res. 1997;25:1935–1942. - PMC - PubMed
    1. Watkins NE, Jr, SantaLucia J., Jr Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes. Nucleic Acids Res. 2005;33:6258–6267. - "VSports注册入口" PMC - PubMed

Publication types

MeSH terms

Substances

"VSports手机版" LinkOut - more resources