Non-specific antibody binding limits the sensitivity of antibody-based detection technologies. We explore the use of logical operations among DNA logic tags associated with antibodies to increase specificity and sensitivity. DNA sequences were developed to perform a logical NOT operation to subtract non-specific binding prior to PCR amplification. Antibody-associated tags A and A' were designed to be partially complementary and contain a restriction enzyme site. Tag A is associated with a specific antibody; tag A' is associated with an isotype matched control antibody. If the concentration of A is greater than A', hybridized AA' is enzymatically cleaved and remaining tag A is subsequently amplified during real-time PCR. Quartz crystal microbalance (QCM), DNA agarose gels, and PCR were used to experimentally characterize components of the NOT operation in respiratory syncytial virus (RSV) detection. QCM showed gold nanoparticles functionalized with both tag DNA and antibody can bind virus. Successful enzymatic cleavage of AA' was visualized on a DNA agarose gel. After cleavage, remaining tag A was amplified by the addition of primers and standard real-time PCR. In the presence of RSV, magnetic pulldown led to the delivery of both tag A and A'. When PCR was run after enzymatic cleavage, the PCR Ct was increased, indicating a decrease in initial tag A. Our results suggest that combining the careful design of DNA logic tags, their association with antibodies, and standard molecular biology techniques is a promising approach to increase the specificity and sensitivity of antibody-based detection methods.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07272007-162434 |
| Date | 03 August 2007 |
| Creators | Vargis, Elizabeth Ann |
| Contributors | F. R. Haselton |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07272007-162434/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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