Self-reporting Microarray Platforms that use
Molecular Beacon Probes
Microarray platforms are widely used because they are able to detect and identify many different nucleic acid sequences simultaneously. Many platforms use fluorescence-based detection techniques that require multiple steps to incorporate fluorophores into target sequences before analyzing them with the microarray. An attractive alternative is to use a platform that contains probes that become fluorescent upon binding to their target. Since molecular beacon probes remain dark when not hybridized to their target, they are especially suitable for use as signaling molecules on selfreporting DNA microarray platforms. In solution-based hybridization assays, molecular beacons have shown high sensitivity and high specificity for target nucleic acid sequences, and they are able to generate fluorescence signals as high as 200-fold greater than their fluorescence background. However, in previous studies, where molecular beacon probes where immobilized on solid surfaces, increases in the fluorescence signals were much lower, often in the single digits, due to high fluorescence backgrounds. The reduced performances are mainly attributed to molecular beacon-surface interactions, which compromise the function of molecular beacon probes.
We developed a novel platform in which molecular beacons are immobilized within highly hydrated microhydrogels, creating a local environment that mimics solution-based hybridization events, and does not compromise the inherently performance of molecular beacon probes.
This work appeared in the March 23, 2012, issue of Soft Matter (Volume 8, Number 11, pages 3067-3076) and the cover of the journal featured our publication.
Recent Publications from our group
Vargas DY, Marras SAE, Tyagi S, and Kramer FR. (2018) Suppression of Wild-Type Amplification by Selectivity Enhancing Agents in PCR Assays That Utilize SuperSelective Primers for the Detection of Rare Somatic Mutations. The Journal of Molecular Diagnostics, 20, 415-427
Schlachter S, Chan K, Marras SAE, and Parveen N (2017) Detection and differentiation of lyme spirochetes and other tick-borne pathogens from blood using real-time PCR with molecular beacons. Methods in Molecular Biology 1616: 155-170.
Catrina IE, Bayer LV, Yanez G, McLaughlin JM, Malaczek K, Bagaeva E, Marras SAE, and Bratu DP (2016) The temporally controlled expression of Drongo, the fruit fly homolog of AGFG1, is achieved in female germline cells via P-bodies and its localization requires functional Rab11. RNA Biol 13: 1117-1132.
Vargas DY, Kramer FR, Tyagi S, and Marras SAE. (2016) Multiplex real-time PCR assays that measure the abundance of extremely rare mutations associated with cancer. PLoS ONE 11, e0156546.
We describe the use of SuperSelective primers that enable the detection and quantitation of somatic mutations whose presence relates to cancer diagnosis, prognosis, and therapy, in real-time multiplex PCR assays that can potentially analyze rare DNA fragments present in blood samples (liquid biopsies), thereby providing information that can be used to modify therapy for individual patients, prolonging (and improving the quality of) life.
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