Single-base mismatches

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. 

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. 

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.

K. Hashimoto and Y. Ishimori, Preliminary evaluation of electrochemical PNA array for detection of single base mismatch mutations, Lab. Chip, 1 (2001) 61-63. 

Fig. 53.4. Histogram that shows the current intensities of DPV peaks obtained for the hybridization responses of 8 pgmL-1 of target associated with cystic fibrosis (T), single-base mismatch (MX-1), three-base mismatch (MX-3), and non-complementary DNA (NC) on magnetic graphite-epoxy composite electrode. Error bars show the mean and the standard deviations of the measurements taken from three independent experiments. Conditions Hybridization temperature, 25°C amount of MB, 100 pg. Other conditions as in Fig. 53.2. With permission from Ref. [3].

M. J. Heller, Rapid Determination of Single Base Mismatch Mutations in DNA Hybrids by Direct Electric Field Control , Proc. Natl. Acad Sci. USA, 94(4), 1119-1123(1997). 

Patolsky F, Lichtenstein A, Willner I (2001) Electronic transduction of DNA sensing processes on surfaces amplification of DNA detection and analysis of single-base mismatches by tagged liposomes. J Am Chem Soc 123 5194-5205... 

Sosnowski RG, Tu E, Butler WF, O Connell JP, Heller MJ. Rapid determination of single base mismatch mutations in DNA hybrids by direct electric field control. Proc Natl Acad Sci USA 1997 94(4) 1119-1123. 

MutS recognizes and binds to single base mismatches and small insertion deletion mispairs (or loops) (Figure 23.12). It can bind mismatches as a dimer or tetramer,... 

Figure 3 Monofunctional (top) and bifunctional (bottom) bulky metallointercalators that target single base mismatches in DNA. In the center is shown a view of the crystal structure of the complex inserted into the DNA from the minor groove at the mismatched DNA site, with ejection of the mismatched bases (14).

Metallointercalators that selectively and efficiently target single base mismatches have found several applications both as biologic probes and as potential chemotherapeutic agents. For instance, [Rh(bpy)2phzi] + was used to probe the relative frequency of mismatched sites in cell lines deficient versus proficient in their mismatch repair machinery (18). The relative cleavage observed with the phzi complex in healthy cell lines was low compared with that in cancer cell lines that carried mutations in essential repair proteins. These results support previous studies on the association of mismatch repair deficiency and cancer. 

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