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| - Comparison between DPO™ primer VS. Non-DPO™ primer |
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M: size marker
Lanes 1: perfect match
Lanes 2: Mismatches at 3'-end
Lanes 3: Mismatches at 5'-end
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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). |
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| 2. Unparalleled high specificity |
| Example 1. DPO™ specificity over a wide range of annealing temperature |
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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™ |
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| 3. 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. |
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| 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. |
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| Seegene has incorporated, or combined the DPO™-Multiplex PCR technology into molecular diagnostics and generates the powerful multiplex diagnostics system (Seeplex™). |
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| 4. Single base discrimination
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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
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Multiple-genotyping
- - Decide genotype and subtype of various pathogens.
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Multiple-SNP Detection
- - Specific and simultaneous analysis of multiple single nucleotide polymorphic (SNP) sites
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| 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.
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- 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
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| 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. |
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| 5. Guaranteed reproducibility |
| High DPO™ specificity with GC-rich template |
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The amplification of the human
klotho target sequence containing 81 %
GC was compared using DPO™ primer and Non-DPO™ primer.
The amplification of the human
klotho target sequence containing 81 %
GC was compared using DPO™ primer and Non-DPO™ primer.
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M: Size marker
Lane 1: Short Non-DPO™ primer.
Lane 2: Long Non-DPO™ primer.
Lane 3: DPO™ |
- 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.
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M: Size marker
Lane 1: Short Non-DPO™ primer
Lane 2: Long Non-DPO™ primer
Lane 3: DPO™ |
