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Our lab has a long-standing interest in in vitro selection techniques; these have included identification of optimal binding sites for antisense oligonucleotides, as well as optimal target sites for ribozymes and DNAzymes in long-target RNAs. Here we have developed methods for identification of DNA aptamers, which reduce and/or eliminate complications caused by fixed flanking primer sequences. The presence of fixed primer flanking sequences can lead to both false positives (when the primer sequences contribute to the binding to the target), which can only be determined by testing the individual random sequences, as well as false negatives (when the primer flanking sequences interfere with binding of the random library sequences). These techniques have been applied in the development of human papillomavirus (HPV), hepatitis B virus (HBV), and other therapeutics, and are now being used for detection of circulating tumor cells and tumor markers. This work is being funded through the National Institutes of Health's Innovative Molecular Analysis Technologies (IMAT) Program.
www.clawsonlab.com
We are involved in the development of a chip-based RNA sensor, which is initially being developed for detection of circulating tumor cells in blood. This sensor uses a “hybridization sandwich” for detection of resonance frequency shifts in nanowires, and sites in target RNAs are optimized using in vitro selection techniques. We are currently expanding this basic platform with the use of aptamers, selected with the protocols described in our article, which will recognize and quantify known tumor markers in plasma. Aptamers will be identified, which recognize different epitopes in known tumor markers, thereby forming an analogous “sandwich,” allowing identification and quantification of the tumor markers in plasma.
See “Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries” on page 351.
