Ribozymes are known to catalyze biochemical reactions and behave like enzymes. They are naturally occurring and have very diverse functions within a cell. After investigating ribozymes that next step was to find if DNA can exhibit the same characteristics since RNA and DNA only differ by a ribose 2'-hydroxyl group. This evolution in curiosity gave rise to artificial DNA enzymes that can catalyze certain reactions and have been created by in vitro selection methods. Due to the ability to manipulate and control DNA hybridization, the deoxyribozyme is advantageous to the field of molecular diagnostics. Other hybridization probes like Taqman for PCR (polymerase chain reaction) or a molecular beacon are more conventional methods for molecular diagnostics, but deoxyribozyme-based nucleic acid sensors are overall more sensitive due to their catalytic enhancement of a signal and more selective due to structural design. When the deoxyribozyme is split into two probes, it is very efficient in identifying a minute difference in sequence compared to the monolith structure. This binary deoxyribozyme sensor (BiDz) has two probes, each containing an analyte binding arm, substrate binding arm, and half of the catalytic core. The monolith structure, known as a catalytic molecular beacon (CMB), contains a hairpin that contains the analyte binding arm in the loop and the substrate binding arms in the stem. The catalytic core is fully intact but deemed inactive due to the substrate binding arms being complimentary to an inhibitory sequence forming the stem. Once the sensor binds the analyte, catalytic core is formed/activated and cleaves a substrate containing a fluorophore and quencher. When the substrate is cleaved a fluorescent signal is given off denoting the detection of the target DNA. Deoxyribozyme sensors can be applied to the field of human sex determination by detecting the Amelogenin gene. Found on both sex chromosomes, the Amelogenin gene is the most common marker used for sex determination because it exhibits dimorphism in length and sequence. Sex identification from ancient skeletal remains is crucial to understanding the social structure of our history. When conventional methods, such as metric analysis, are not an option due to the fragmented or prepubescent remains, molecular diagnostics are needed. Amplification of DNA is required to be able to detect the target sequence in human samples. Isothermal loop-mediated amplification (LAMP) is a fast and simple technique that provides ample amounts of amplicon. It is advantageous over PCR because it amplifies at one temperature and no thermal cycler is needed. Two different sensors have been designed to detect the X and Y specific sequences with high selectivity. From a direct comparison between the CMB and BiDZ, the binary structure has shown to be simpler and less expensive to design, and highly selective toward single base substitutions (SNS). While both sensors contain detection limits in the picomolar range, which is consistent with data published by other research groups, the CMB sensors failed to function at higher temperatures (55oC). BiDz sensors are shown to be superior to the CMB design, particularly when selectivity based analysis is desired. For human sex determination, the binary sensor detected sex specific sequences with great selectivity. The sensor then detected LAMP amplified DNA from male and female teeth after 30 minutes of amplification. Combining a binary deoxyribozyme sensor and isothermal amplification can provide a new and valuable method for human sex determination.
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Doctor of Philosophy (Ph.D.)
College of Sciences
Length of Campus-only Access
Doctoral Dissertation (Campus-only Access)
Smith, Alexandra, "A Deoxyribozyme Sensor and Isothermal Amplification for Human Sex Determination" (2017). Electronic Theses and Dissertations. 5931.