Base mismatch

The short length of a typical ASON facilitates cell internalization and increases hybridization efficiency by reducing base-mismatch errors. Once hybridization has occurred the ASON-mRNA complex becomes a substrate for intracellular RNAses (e.g., RNAse-H) that catalyze mRNA degradation and allow ASON to recycle for another base pairing with the next target mRNA molecule. The net result of this process is a sustained decrease of target mRNA translation and a lower intracellular level of the corresponding protein (Fig. 1). 

To test this sensitivity of photoinduced quenching to perturbations in the intervening base pair stack, an assembly was prepared containing an intervening CA base mismatch which disrupts locally the /r-stack between the donor and acceptor. We observed that the yield of CT was reduced in the DNA... 

Considerable amounts of quenching of the acridone emissions by guanine in the DNA occurred when guanine was close to acridone, which can be applied as a quencher-free probe (no additional quencher is required) for the detection of a special sequence of DNA. The DNA bearing acridone at the C5 position of inner thymidine could distinguish the opposite T-T base mismatch, while enhancement of discrimination ability is needed for the practical use of single nucleotide polymorphism (SNP) typing. 

Liebermann T, Knoll W (2003) Parallel multispot detection of target hybridization to surface-bound probe oligonucleotides of different base mismatch by surface-plasmon field-enhanced fluorescence microscopy. Langmuir 9 1567-1572... 

The acridinium ester (AE) in an AE-labeled cDNA probe hybridized to target DNA is less likely to be hydrolyzed than in the unhybridized conformation (Fig. 10) [9-11]. Single-base mismatches in the duplex adjacent to the site of AE attachment disrupt this protection, resulting in rapid AE hydrolysis [11]. Hydrolysis by a weak base renders AE permanently nonchemiluminescent. After hydrolysis, it is possible to use the remaining chemiluminescence as a direct measure of the amount of hybrid present. This selective degradation process is a highly specific chemical hydrolysis reaction, which is sensitive to the local environment of the acridinium ester. The matched duplex can be detected and quantified readily, whereas the mismatched duplex produces a minimal signal. 

All DNA hybridization assays are subject to cross-hybridization, in which an oligonucleotide that is not a perfect sequence match hybridizes with the capture probe. The cross-hybridization in the OFRR was investigated using samples of oligonucleotides with either 0-, 1-, 2-, 5-, or 25-base mismatches when compared with the 25 base-pair biorecognition capture probe. The resulting resonant mode spectral shifts for the respective mismatch are plotted in Fig. 14.7b. The measurements show a difference of 1.3 pm and 2.8 pm for one and two base-pair... 

Fig. 10 (a) Photos of colorimetric changes in solutions of (7.9 x 10 5 M, on a monomer unit basis) (b) a) polymer b) polymer/DNA probe c) polymer/DNA probe/complementary DNA sequence d) polymer/DNA probe/1 base mismatched DNA sequence e) polymer/DNA probe/ 2 base mismatched DNA sequence after 5 min mixing at 55 C in 0.1 M NaCl/H20. B) UV-vis spectra of the above corresponding samples [19]... 

In CSGE, mildly denaturing solvents in an appropriate buffer can accentuate conformational changes produced by single-base mismatches in heteroduplexed DNA. This increases the differences in electrophoretic mobility between heteroduplex and homoduplex. 

Discrimination is seen over a wide interval of concentrations (Fig. 26.7). Signals are linear for both complementary and one-base mismatched strands, between 0.01 and InM. Detection limits of 5 and 70 pM were, respectively, obtained. 

Fig. 26.6. Results obtained for immobilised and complementary (c-DNA) or one-base mismatched (lm-DNA) target strand hybridisation, combining different lengths. Ctarget = 2.5 nM, Vtarget = 20 pL, 2 x SSC pH 7 buffer with 50% formamide, thybr = 60 min. Data are given as average+SD (n — 3).

Fig. 26.8. Schematic representation of the analytical procedure followed for the construction of the genosensor and the detection of a complementary target and a single-base mismatch target. (A) Electrocatalytic and (B) enzymatic detection.

The ply genosensor has been used for detecting oligonucleotide sequences containing a one- or three-base mismatch. Three different... 

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