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DPO™ technology is a fundamental tool for blocking extension of non-specifically primed templates generating consistently high specificity. The strength and utility of this DPO™ technology can be successfully incorporated into molecular diagnostics systems such as multiplex diagnostics and SNP genotyping systems.
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 through considerable time and effort are devoted to primer design and optimization of reaction conditions.
A novel DPO™ system that is structually and functionally different from the primer system currently in wide-spread use blocked extension of non-specially primed templates, and thereby generates consistently high PCR specificity even under less optimal PCR conditions.
Super Multiplex PCR
1. Freedom in primer design & PCR optimization
2. Unparalleled high specificity
3. No primer competition and dimerization in Multiplex PCR
4. A wide variety of application
5. Guaranteed reproducibility
 
1. Freedom in primer design & PCR optimization
DPO™ comprises of two separate priming regions (a first priming region and a second priming region) joined by a polydeoxyinosine linker. The linker forms like a "bubble-like structure" which itself is not involved in priming, but rather delineates the boundary between two parts of primer.
- Principle of DPO™
DPO™ has two functional prming regions (one is longer than the other) separated by the poly (I) linker. These two unequally distributed priming regions generate dual priming reactions based on the following scheme, resulting in only target-specific products.
Step 1: Poly(I) linker activation
Deoxyinosine has a relatively low melting temperature compared to the natural bases, due to weaker hydrogen bonding so that the poly (I) linker will form a bubble-like structure at a certain annealing temperature and separates a single primer into a two functional regions.
Step 2: First priming reaction
The longer 5'-segment preferentially binds to the template DNA and initiates "stable annealing". It acts as a Stabilizer".
Step 3: Second priming reaction
The short 3'-segment selectively binds to a target site and determines "target-specific extension". It acts as a "Determiner".
- Comparison between Conventional primer VS DPO™
M: size marker
Lanes 1: perfect match
Lanes 2: Mismatches at 3'-end
Lanes 3: Mismatches at 5'-end
Fig. 1.Comparison of Ndufs2 products obtained using conventional primer and DPO™

Another major advantage is that DPO™ makes primer design extremely simple and easy Since two separate priming reactions provide a primer with a comfort zone (high tolerance) in annealing. Following the first stable priming reaction by Stabilizer, the second critical priming reaction by Determiner gives one additional chance to correct the specificity. For this reason, DPO™ does not require a rigid optimization of PCR conditions and primer search parameters including primer length, GC content, annealing temperature, and secondary structure (hairpin, self or cross dimer).
2. Unparalleled high specificity
Example 1. DPO™ specificity over a wide range of annealing temperature
The conventional primers reduced the non-specific products by increasing the annealing temperature to 65 ℃ (lanes 1 and 3), while the DPO™ primer produced only one target product over a wide range of annelaing temperatures (55 ℃ and 65 ℃) (lanes 2 and 4).

M: Size marker
Lane 1: Conventional primer
Lane 2: DPO™
3. No primer competition and dimerization in Multiplex
Multiplex PCR is a rapid and economical tool, but when a large bank of genes are amplified with multiple-primers sets, conventional primers often produce false positives due to primer competition, to primer dimers or to the different melting temperatures of the different primers, Therefore current multiplex PCR-based assays require further validation, such as nested PCR, or a probe hybridization assay. However, DPO™ allows specific detection of a large number of pathogens without any false result because the bubble-like structure of the poly(I) linker in DPO™ efficiently prevents primer-dimer and hairpin structure formation. DPO™-Multiplex PCR generates the high specificity without production of any non-specific bands or false-positive products and it represents a reliable, rapid, practical and cost-effective detection method.
Seegene has incorporated, or combined the DPO™-Multiplex PCR technology into molecular diagnostics and generates the powerful multiplex diagnostics system (Seeplex™).
4. Single base discrimination
Multiple-pathogen Detection
  • - High specificity without production of any non-specific or false-positive results
    - Reliable, rapid, practical and cost-effective detection method
    - Specific and simultaneous detection of multiple pathogens without any false results
 Multiple-genotyping
  • - Specific and simultaneous discrimination of multiple pathogen subtypes with similar genetic background
Multiple-SNP Detection
  • - Specific and simultaneous analysis of multiple single nucleotide polymorphic (SNP) sites
  
Single nucleotide polymorphisms (SNPs) are responsible for drug resistance leading to various diseases and make diagnosis difficult. In general, conventional primers cannot offer reliable results for SNP genotyping and current SNP genotyping methods requires additional steps after amplification of an SNP-containing region such as RFLP, sequence or hybridization. There are neither rapid nor easy to manipulate, and they require large initial investments in equipment. In contrast, DPOTM-SNP detection PCR makes it possible to achieve SNP genotyping in one PCR step.
  • Hetero type of allele 1 & 2: lanes 1~3
  • Homo type of allele 1: lanes 4~6
  • Homo type of allele 2: lanes 7~9

SNP in CYP2C19 results from a single base pair substitution (G→A) at position 681 in exon 5 of CYP2C19 and causes a non-functional protein and affects the metabolism of a number of commonly used drugs1).

Multiplex PCR analysis of nine human genomic DNA samples with known genotypes at the CYP2C19 locus using conventional primers (left) and DPO™ (right) was carried out. As a result, DPO™-based multiplex PCR clearly distinguished between the different alleles of CYP2C19, while conventional primer-based multiplex PCR did not.
5. Guaranteed reproducibility
High DPO™ specificity with GC-rich template
The amplification of the human klotho target sequence containing 81 % GC was compared using conventional primers and DPO™.
The amplification of the human klotho target sequence containing 81 % GC was compared using conventional primers and DPO™.
M: Size marker
Lane 1: Short conventional primer
Lane 2: Long conventional primer
Lane 3: DPO™
Amplification of the human GSPT1 target sequence containing 81 % GC with conventional primers and DPO™.
A. PCR reaction excluding the detergent (5% DMSO + 1M Betaine).
The conventional primers generate many non-specific products in a PCR reaction omitting the detergent, whereas the DPO™ does not amplify any PCR product.
B. PCR reaction including the detergent.
When the detergent are added to relax secondary structures for higher GC content, DPO™ successfully amplifies PCR product.
M: Size marker
Lane 1: Short conventional primer
Lane 2: Long conventional primer
Lane 3: DPO™
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