Rrest was attributed to DNA polymerase’s inability to displace the 5′ end of the duplex superstabilized with the CDPI3 group. It has since been shown that 5′-CDPI3-ODNs are able to arrest Taq DNA polymerase and therefore can be used at PCR temperatures as well. This came as a surprise since it was expected that the 5′ exonuclease Taq polymerase activity would degrade such duplexes. Evidently, 5′-CDPI3 labeling makes ODNs resistant to 5′ exonuclease digestion. Such ODNs can be used as PCR blockers to prevent amplification of selected DNA sequences.
FIGURE 3: MECHANISM OF ACTION OF FLUOROGENIC CDPI3-FLUOROPHORE-PRIMERS
F
PETquenching CDPI3
-3′-OH’
Primerextension
DNAtarget 2. Short and fluorogenic PCR primers Efficient priming of PCR was demonstrated with 5′-CDPI3-ODNs as short as 8-mers using modified (touch-down) PCR cycling conditions or 10-16-mers using regular PCR cycle.155270-99-8 MedChemExpress 7 The PCR was shown to produce specific amplification products of expected size. The reduced length primers were suggested for use for PCR amplification of viral sequences which possess a high degree of variability or techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.19545-26-7 site 5′-CDPI3-primers which also have an attached 5′-fluorophore are able to quench the dye fluorescence via the photo-induced electron transfer (PET) mechanism, depicted in Figure 3. Such primers are significantly quenched in a single strand state but become highly fluorescent when incorporated into the PCR amplicon allowing for detection of target amplification.PMID:25905250 3. Real-time PCR probes The stronger binding of CDPI3-ODNs in comparison with unmodified ODNs allows for more stringent hybridization conditions to be used in DNA hybridization assays. Short CDPI3-ODNs with improved mismatch discrimination are especially useful in PCR assays since they bind efficiently and specifically during the high-temperature primer extension cycle. Several types of real-time PCR probes that utilize the CDPI3 moiety have been developed. MGB TaqManprobes4 have CDPI3Quencher and Fluorophore tethered to the 3′ and 5′ ends, respectively. Provided that specific sequence is present in the target DNA, the TaqMan probes are degraded by Taq polymerase during PCR, releasing unquenched fluorophore. MGB Eclipsehybridization probes8 have the CDPI3-Quencher and Fluorophore attached at the 5′ and 3′ ends, respectively. They are non-degradable and their fluorescence is strongly increased upon
probes’ hybridization to amplified targets during the annealing step. MGB Pleiadesprobes9 are also nondegradable hybridization probes. They have the CDPI3-Fluorophore and Quencher moieties tethered to the 5′ and 3′ ends, respectively. These probes utilize the unique dual fluorescence quenching mechanism to significantly reduce background fluorescence and improve signal-to-background ratio.
2002, 25, 1-8. [9] E.A. Lukhtanov, S. Lokhov, V.V. Gorn, M.P. Podyminogin, W. Mahoney Nucleic Acids Res. 2007; 35, No. 5 e30,doi:10.1093/nar/ gkl1136. [10] A. Khvorova, A. Vermeulen, R. Kaiser, J. Karpilow, N. M. J. Vermeulen, W. Mahoney US9334495 Minor groove binder (MGB)oligonucleotide miRNA antagonists [11] D. L. Mcelligott WO2012119051 Enhanced biodistribution of oligomers
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