Improved DNA hybridization assays using surfactant probes.

摘要

Detection of nucleic acids is important for applications such as identification of various microorganisms, tracking levels of cellular mRNA, and gene expression. Present day RNA/DNA detection methods include microarrays, reverse transcriptase polymerase chain reaction (PCR) and real time PCR using fluorescent probes. They provide information on the type and amount of RNA present. Although sensitive, these methods are very time consuming, labor intensive, and require calibration with known concentrations of pure RNA. RNA can be detected either directly from cellular lysates containing various cellular macromolecules such as proteins and lipids, or indirectly detected as its DNA form (cDNA), a PCR product. Microarrays are limited to pure RNA or cDNA samples due to potential surface fouling and probe deactivation by cell lysate components. Although RNA can be measured from cell lysates using PCR with molecular beacons or TaqMan fluorescent probes, it is time consuming and any variations induced by the operator and reagents will be amplified during PCR cycles. Reverse transcription PCR of cDNA is sensitive and provides higher detection limit of RNA; however, RNA detection from cell lysates decreases analysis time.;We develop a solution-based detection of nucleic acids via their separation in micellar electrokinetic chromatography (MEKC) by hybridization to surfactant probes. The major application we envision is direct detection of raw RNA; however, the same method can be extended to a PCR amplified cDNA with a covalently attached surfactant probe. We will show that RNA detection via a surfactant probe in MEKC is advantageous over microarrays and fluorescent probes in PCR due to its simplicity and fast analysis time. When compared to microarray detection that takes up to 3 days or PCR that takes 4 -- 5 hours, MEKC detection takes about 30 minutes including sample preparation. In addition, we are able to obtain information on RNA length. This increases the confidence in RNA identification.;We investigate the impact of unhybridized RNA orientation (overhanging part of RNA that is not hybridized to the probe) and serum biological fluid components on the detection and separation efficiency in MEKC. A surfactant probe contains an alkane tail and the separation is achieved via differences in hydrophobicity of the probe. We utilize those differences to obtain multiple RNA detection simultaneously. This method provides an accurate and fast detection of target RNA and avoids limitations associated with surface based detection via microarrays and solution based detection using fluorescent probes in polymerase chain reaction (PCR).;Two surfactant probes are studied in this project. One is a peptide nucleic acid amphiphile (PNAA) which is a modified peptide nucleic acid where an aliphatic tail and amino acid residues are attached to promote hydrophobicity and better solubility for the use in MEKC. The second probe is an alkylated DNA (aDNA) where a hydrophobic tail is attached via amide bond linkage. Upon probe-RNA hybridization, a hydrophobic tail promotes target RNA interaction with micelles via partitioning into the interior of Triton X-100 (TX-100) micelles used in MEKC. The extent of partitioning is dependent on the degree of probe hydrophobicity and concentration of micelles present. Thus, target RNA is separated and detected from the pool of nucleic acids. After careful investigation of PNAA and aDNA we conclude that aDNA is a better suited probe for RNA detection due to its fast and simple synthesis, higher hybridized duplex stability, and reduced interaction with serum contaminants. Due to chemical similarities between RNA and DNA, we did preliminary studies in this thesis using easily obtained synthetic DNA oligomers for experimental convenience. (Abstract shortened by UMI.)