Mismatch detection

Figure 10 Mismatch detection by using a chemiluminescent AE-labeled cDNA probe. Procedure [9, 11] Acridinium ester-labeled probes specific for either wild-type or mutant sequence corresponding to a target DNA are hybridized with the sample DNA for 1.0 h at 60°C in a hybridization buffer (pH 5.2). Hybridized and nonhybridized probes are discriminated by the hydrolysis reaction for 12 min at 62.5°C in the presence of Na2B407 (pH 8.5) and Triton X-100. The chemiluminescence of each sample is then measured in a luminometer.

PNA strand, resulting in an increase of the electrochemical signal (SWV peak current) as a result of probe-target hybridization. The PNA-functionalized conductive polymer sensor allowed for a detection limit of approximately 10 nM, and the feasibility for single-nucleotide mismatch detection was also demonstrated. 

Dubertret B., Calame M., and Libchaber A. J. (2001). Single-mismatch detection using gold-quenched fluorescent oligonucleotides. Nat. Biotechnol 19 365-370. 

For example, the light switch intercalator complex [Ru(phen)2dppz] (Fig. 4.19) is nonemissive in the presence of single-stranded DNA but emits brightly in the orange-red in the presence of double-stranded DNA, which provides a much better intercalative platform [306]. This complex has been covalently attached to the 5 ends of oligonucleotides, and mismatch detection at room temperature has been achieved based on the increase of Ru(II) complex emission with an increased double-stranded character [306] (Fig. 4.28). The presence of one mismatch 1 of 15 led to a loss of one-third to two-thirds of the emission intensity of the perfectly hybridized duplex, depending on mismatch location [306]. 

Figure 430. Color-coded spot test for DNA mismatch detection with gold nanoparticles, showing the red/blue color changes. [From Fig. 7 of Ref. 311, with permission.] (See color plates).

Fig. 2.7 Schematic of the DNA base-pair mismatch detection system based on an Os redox polymer. (Adapted from [81])...

In order to be useful for the detection of nucleotide basepair mismatches, the electrochemical signature of the mismatched ds-ODN must be significantly different from that of the fully hybridized ds-ODN. In this chapter, we summarize the state of the art in this field and provide an overview over capture strand immobilization strategies and various mismatch detections schemes. 

Numerous electrochemical strategies have been developed for the detection of mismatches in DNA. These vary from the use of electroactive DNA intercalators to enzymatic signal amplihcation schemes, or redox-modified oligonucleotides. In the following sections, we will focus on the discussion of a range of electrochemical mismatch detection schemes. 

For example, it was demonstrated by Barton [32] that doublehelical DNA films on gold surface display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Moreover, it has been observed that mismatch detection is possible regardless of DNA sequence composition and mismatch identity. The presence of mismatches was elucidated based on the electrochemical characteristics of the redox active intercalators bound to the DNA-modified gold surfaces. Coupled redox reactions were employed to induce an electro-catalj ic current and thus increase the method s sensitivity (Fig. 7.4). 

FefCN] ] " by methylene blue (MB] at a DNA-modified electrode. LB+ is leucomethylene blue, the product of the electrochemical reduction, (b] Cyclic voltammetry at a gold electrode modified with DNA of 2 mM [Fe(CN]6] (curve 1], 2 p.M MB (curve 2], and 2 mM [FefCNJe] and 2 p.M MB (curve 3]. Reproduced from S. 0. Kelly, E. M. Boon, J. K. Barton, N. M. Jackson, and M. G. Hill, Single-base mismatch detection based on charge transduction through DNA, Nucleic Acids Research, 1999, 27(24], 4830-4837, by permission of Oxford University Press. 

There are a number of reports of mismatch detection strategies, in which enzymatic reactions are exploited to amplify the electrochemical signal. 

For practical application, an ideal biosensor must be as straightforward as possible with least number of synthesis and analytical steps. Kraatz and coworkers introduced a simple, label-free and sensitive electrochemical sensor for single nucleotide mismatch detection. This approach relies on the diffusive property of the negatively charged redox probe [Fe(CN)6] and its interplay with matched and mismatched DNA films. 

Hybridization and one-base mismatch detection was performed by using self-assembled monolayer (SAM) on gold electrodes in the presence of MB indicator first time [61]. 14-mer short oligonucleotides were immobilized onto Au electrode surface by using alkanethiol monolayer coupling at surface. Mercaptopropionic acid (MPA) was used for monolayer production. Voltammetric reduction signal of MB was monitored for hybridization and mismatch detection. 

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