Η μικροτεχνολογία στην ανάλυση DNA και RNA

Abstract

In recent years there is a continuously increasing research activity towards the development of miniaturized analytical devices (chips) for detection and/or determination of nucleic acids. The interest for miniaturization arises from the increase of the speed of analysis, decrease of the cost, analysis of thousands of sequences in parallel (multianalyte systems) and the possibility of integration of multiple analytical steps into a single microdevice. The research activity described in this dissertation lies at the interface between modern (bio)chemical instrumentation and analytical chemistry of nucleic acids. The Theoretical Part of the dissertation (Chapter 1) starts with a description of polymerase chain reaction (PCR), the most widely used technique for exponential amplification of nucleic acids. The basic principles of DNA separation by gel electrophoresis and capillary electrophoresis are then presented. Also, this Chapter provides an introduction to nucleic acid hybridization assays, a discussion about multianalyte assays and microarrays as well as a presentation of the reporters, radioactive and non-radioactive. Next, various systems for in vitro expression (protein synthesis) as well as the principles of molecular design of DNA templates suitable for in vitro expression are presented. Finally, the properties and chemical reactions of bioluminescent proteins are discussed. Chapter 2 (Experimental Section) describes the development of a home-built integrated microarray system (IMAS) comprising a three-laser confocal fluorescence scanner coupled with a microarray printer. Microarray technology covers the urgent need to exploit the accumulated genetic information from large scale sequencing projects and facilitate investigations on a genome wide scale. Although most applications focus on DNA microarrays, the technology has expanded to microarrays of proteins, peptides, carbohydrates and small molecules aiming either at detection/quantification of biomolecules or investigation of biomolecular interactions in a massively parallel manner. Microarray experiments require two specialized instruments: An arrayer (or printer) for construction of microarrays and a readout instrument (scanner). We have designed, constructed and characterized the first integrated microarray system (IMAS) that combines the functions of a microarrayer and a three-laser confocal fluorescence scanner into a single instrument and provides excellent flexibility to the researcher. The three-axis robotic system that moves the printing head carrying multiple pins for arraying is also used for moving the microarray slide in front of a stationary optical system during scanning. Since the translation stages constitute the most expensive and crucial components of microarray printers and scanners, the proposed design reduces considerably the cost of the instrument and enhances remarkably its operative flexibility. Experiments were carried out at resolutions of 2.5, 5, 10 and 20 μm. The scanner detects 0.128 nM carboxyfluorescein (spots with diameters of 70 μm) corresponding to 1.8 molecules/μm2. The linear range extends over 3.5 orders of magnitude (R2=0.997) and the dynamic range covers 4.89 orders of magnitude. DNA microarray model experiments were carried out including staining with SYBR Green I and hybridization with oligonucleotides labeled with the fluorescent dyes Alexa 488, Alexa 594 and Alexa 633. The objective of Chapter 3 is the molecular assembly of DNA in a microfluidic device. We used a microfabricated device that performs continuous-flow PCR. In the device, the DNA template was amplified with a concomitant fusion of the appropriate sequences that enable in vitro transcription and translation of the DNA products. As model templates for PCR, we used DNA molecules encoding the photoprotein aequorin (Aeq, MW = 22000) and the enzyme firefly luciferase (FLuc, MW = 62000). The Aeq and FLuc DNA were subjected to PCR in the chip using primers containing the T7 RNA polymerase promoter, Kozak sequence and the start codon (ATG). Aeq and FLuc PCR products, 650 bp and 1720 bp, respectively, were introduced into an in vitro expression system (based on wheat germ) and the activity of generated proteins was measured based on their bioluminescent reaction. The combination of on chip PCR with in vitro expression was used as a sensitive approach to investigate amplicon carry-over between successive PCRs and for the study of the reproducibility of amplification. It was shown that the amount of generated protein is a function of the initial number of DNA template molecules introduced in the PCR chip. The entire procedure is complete in 60 min and requires only 1 mL for PCR and 3 mL for expression. Furthermore, we achieved complete reassembly of digested DNA molecules in the microfluidic device. A DNA fragment encoding apoaequorin was digested with DNase I and the products were separated by gel electrophoresis. Fragments ranging from 50-250 bp were isolated from the gel and served as templates for assembly PCR in the microfluidic device. The PCR product was then introduced into a second PCR on the chip, to generate expressible DNA fragments. The final product was subjected to combined in vitro transcription and translation to produce active aequorin molecules, thereby proving the perfect reassembly of functional DNA molecules in the microfluidic device. Chapter 4 focuses on the development of a reusable continuous-flow capillary electrophoresis chip. Glass microscope slides are used as substrates. The chip was fabricated by using photolithographic and wet chemical etching techniques. The device contains a channel that allows continuous flow of DNA samples (PCR products). Separation takes place using two channels in a cross-type configuration. The chip can be fabricated in two days and the materials cost less than 1 Euro. The separated DNA fragments are detected by laser-induced fluorescence. The system was applied to rapid separation of DNA markers (fragment sizes between 72 and 1353 bp) and PCR products. Separation of the markers was complete within 40 s compared to 20 min and 40 min required for separation by conventional capillary electrophoresis and gel electrophoresis, respectively.