Method development and optimization for nucleic acid detection platforms

Official URL: https://risc01.sabanciuniv.edu/record=b2486373 _(Table of contents)

The nucleic acid tests abbreviated as NAT, is a technique requires amplification and detection to provide guidance on the diagnosis of genetic materials. Although the genetic material of every living consists of DNA or RNA, there are variations in genome sequences. This genetic variation makes NAT an ideal technique for identifying, genetically modified organisms (GMOs), infectious diseases, cancer, genetic disorders, and mitochondrial disorders, helping to improve diagnostic technologies. Nucleic acid amplification requires a laboratory environment with special equipment and technical expertise. Loop Mediated Isothermal Amplification (LAMP) is technically simpler than Polymerase Chain Reaction (PCR). LAMP has ideal properties for nucleic acid detection applications. LAMP assays are robust and has ability of pyrophosphate production in the presence of target, which enables detection with naked eye. Polymerase inhibitors in samples do not affect the amplification process. Most importantly, LAMP makes the reaction suitable for simple targetresponse diagnostic systems with simplified sample preparation. In this thesis, LAMP was primarily developed and optimized according to highlight the strong diagnostic aspects of detection platforms, and their effects on healthcare and its benefits to society. The systems we worked on enlarges the target DNA using LAMP method. In less than 30 minutes, it reacts with pH-dependent dyes (such as hydroxynaphtol blue (HNB)) and enables colorimetric DNA detection with naked-eye. Detection of DNA fragments were performed parallelly in thermal cycler and our platforms. Results show LAMP is an advantageous method because it is highly sensitive, cheap, user-friendly, and safe; in addition, does not usually require DNA extraction (in colony-LAMP). The LAMP reaction is believed to be a simple and reliable tool for laboratory purposes because it needs only very basic instruments and the results can be observed and contrasted visually