DPO™ Technology

Novel Oligo platform of super multiplex PCR

New primer technology that inhibits combined nonspecific template.

Proper priming between oligonucleotide primer and template is important to obtain accurate result from PCR. Dual Priming Oligonucleotide(DPO) primer inhibits occurrence of PCR product (mismatched priming, non-specific priming). A novel DPO™ system that is structurally 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.




Features of the technology

Freedom in primer design & PCR optimization

DPO™ comprises of two separate priming regions (5'-end stabilizer and 3'-end determiner) 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 5'-end stabilizer and 3'-end determiner.

디자인할 수 있는 부위가 자유롭고 PCR 성능을 높여줌

Principle of DPO™
DPO™ has two functional priming 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 Tm value 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 5'-end stabilizer and 3'-end determiner as "stabilizer" and "determiner"

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 DPO™ primer VS. Non-DPO™ primer

Fig. 1.Comparison of Ndufs2 products obtained using DPO™ primer and Non-DPO™ primer

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 of 5' end, 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).

Comparison between DPO™ primer VS. Non-DPO™ primer

Unparalleled high specificity

Example 1. DPO™ specificity over a wide range of annealing temperature

annealing 온도 변화조건에서 특이성을 보여주는 DPO™ Fig. 2. Fig 2. Non- DPO™ primer shows specificity at high specific annealing temperature, and production of false product such as non-specific band occur at low annealing temperature while the DPO™ primer produced only one target product over a change of annealing temperature. (lanes 2 and 4).

M: Size marker
Lane 1: Non-DPO™ primer
Lane 2: DPO™

No primer competition and dimerization in Multiplex

DPO™ technology has high specificity without false-positive. DPO™ Multiplex system using this technology is an accurate, quick, and cost-effective molecular diagnostic method.

Multiplex-PCR 수행시 primer간의 dimer 형성 또는 competition이 없음
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



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
- Decide genotype and subtype of various pathogens.

Multiple-SNP Detection
- Specific and simultaneous analysis of multiple single nucleotide polymorphic (SNP) sites

Occurrence of Single nucleotide polymorphisms (SNPs) are responsible for drug resistance leading to various diseases, but additional steps after amplification of an SNP-containing region such as RFLP, sequence or hybridization are needed to identify SNP genotyping accurately. As a result, the longer time will take for SNP genotyping and additional test will be done. High specificity of DPO enables SNP genotyping with only a single PCR by identifying SNP accurately.

단일 염기 변이 구별 (다양한 적용 범위)

  • 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 (681 bp: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 drugs. 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.

Guaranteed reproducibility

High DPO™ specificity with GC-rich template.

GC contents가 높은 조건에서 specificity를 보이는 DPO™ Fig. 1.The amplification of the human klotho target sequence containing 81 % GC was compared using DPO™ primer and Non-DPO™ primer.

Amplification of the human GSPT1 target sequence containing 81 % GC with DPO™ primer and Non-DPO™ primer.

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.
home > Introduction > Core technology > DPO™ Technology