Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film

Abstract

We developed a new technique to immobilize a set of molecular beacons on an agarose film-coated slide and found that it has the ability to identify a single nucleotide difference in label-free DNA targets. The annealing properties, specificity and hybridization dynamics of the present technique were compared with those of the conventional technique that directly immobilizes molecular beacons on a planar glass slide. It is demonstrated that the molecular beacon array on an agarose film has high quench efficiency, an excellent discrimination ratio for single nucleotide mismatches and a short detection time. We hypothesize that such a low fluorescence background and high specificity molecular beacon array will find practical applications in label-free, high-throughput mutation analysis and disease diagnosis.

Peptide nucleic acids on microarrays and other biosensors

“…This enables the usage of PNA beacons under conditions that are not feasible for DNA beacons. Immobilization on both flat surfaces [27] and optical fibers [28] has been reported.…”

Target discrimination by surface-immobilized molecular beacons designed to detect Francisella tularensis

“…Tyagi and Kramer (1996) described a novel DNA molecular construction referred to as a molecular beacon (MB) that utilizes a novel design of fluorescence energy transfer, and is a good candidate for use at a pathogen-detector interface. MBs can recognize and report the presence of specific nucleic acids in homogeneous solutions, and more recently have been incorporated on various immobilizing platforms (Fang et al, 1999;Liu and Tan, 1999;Brown et al, 2000;Liu et al, 2000;Steemer et al, 2000;Broude et al, 2001;Riccelli et al, 2001;Frutos et al, 2002;Wang et al, 2002). Typically, molecular beacons are 15-25 nucleotides in length that energetically favor forming a hairpin (e.g.…”

“…Microarrays allow multiplex processing, and simultaneous screening of thousands of unique nucleic acid fragments, and possibly may be adopted into a real-time reusable detector format. Researchers have made progress toward surface immobilization of molecular beacons on a microarray format using biotin-avidin coupling (Fang et al, 1999), or aldehyde coupling of free amino groups (Broude et al, 2001;Wang et al, 2002). Approaches have been proposed to improve immobilized molecular beacon sensitivity by increasing the number of probes bound to the glass slide by first treating the slides; for example, treatment by photoor persulfate-lithographic linked acrylamide-containing gels (Vasiliskov et al, 1999), dendrimeric linker systems (Beier and Hoheisel, 1999), or agarose films (Broude et al, 2001;Wang et al, 2002;Afanassiev et al, 2000).…”

Biosensor Recognition Elements

“…Molecular beacon aptamers have been made against clinically relevant molecules such as thrombin (Nutiu and Li, 2004). Of clinical relevance in diagnostic screening is the fact that it has been estimated that 60% of all humans will have been affected by gene mutations in their lifetime and biorecognition elements capable of differentiating between two target DNA sequences differing by only a single nucleotide (Wang et al, 2002) will contribute significantly to high-throughput mutation detection. Additionally, signaling aptamers have the ability to indicate the presence of non-nucleic acid analytes such as proteins and small organic compounds (Rajendran and Ellington, 2002) which make them very useful for general detection purposes.…”