Browsing by Subject "point of care diagnostics"

Nucleic acid amplification technologies are widely used in the field of molecular diagnostics and biological research for amplifying and detecting specific nucleic acid sequence. Polymerase chain reaction (PCR) is the most established nucleic acid amplification technology providing a fast, sensitive and highly specific method to amplify nucleic acids. But, as a "point-of-care" - diagnostic tool, PCR still remains limiting due to the need to use complex and heavy instrument. This disadvantage of PCR has lead to development of nucleic acid amplification technologies, which are possible to utilize with a simple and cost-effective device. PCR requires thermocycling between three different temperatures, while these isothermal technologies are possible to use in a constant and low temperature. Over ten different isothermal nucleic acid amplification technologies are described in the literature within past two decades. This literature review is focused on six of these existing isothermal technologies. They are briefly described and their potential applications to point-of-care diagnostics are discussed. The experimental part of the thesis focuses on improving the sensitivity, speed and robustness of isothermal nucleic acid amplification, SIBA technology. This was achieved by investigating the optimum configuration of the invasion oligonucleotide (IO) required dissociation of target DNA duplex. The invasion oligonucleotide contains of three functionally district regions; the seeding-, DNA- and a 2'-O- methyl RNA region. The IO can also contain a few non-cognate bases at the 3'end, inverted dt. It was researched how the length of the seeding- and DNA regions, and the ratio of pyrimidine in the seeding region affect to the amplification speed and specificity. SIBA reaction was tested also with IOs where the 2'-O- methyl RNA bases of the IO were partially replaced by 2'-fluoro RNA bases and the inverted dT removed. The results revealed that a higher ratio of pyrimidine in the seeding region speed up the nucleic acid amplification reaction, but too high ratio can also induce non-specific amplification. The optimal length of the seeding region lies somewhere between 3 and 18 bases. The optimal length of the DNA region is around 35 bases. This study also showed that the inverted dT blocker of the IO is unnecessary in SIBA, and by replacing 2'-O- methyl RNA with 2'-fluoro RNA bases is possible to improve the amplification of target template with mutations. Results presented in this thesis have helped to improve existing knowledge of SIBA-assay design. Future work could focus on investigating the impact of the IO's 2'-O- methyl RNA region.