Arrayed Primer Extension-2 as a multiplex PCR-based method for nucleic acid variation analysis: method and applications

Human DNA is studied for two main reasons: scientific and clinical interest. In both cases, a robust, cost-effective, and reliable (>99.5%) method of analysis is required. There are several protocols for genetic analysis which enable one to determine DNA variations relatively simply and do not demand a specific apparatus. More complex analysis solutions are provided by commercial companies. They facilitate high-throughput DNA analysis and provide a wider application spectrum compared to non-commercial methods, but the cost per base pair of DNA analyzed is higher. Aside from numerical quality parameters, the essential indicators of a method are flexibility, amount of hands-on work, and simplicity in the starting phase. In conclusion, there are several available platforms but due to the complexity of DNA variations, no dominating methods which can satisfy all research or diagnostic conditions and users exist. The aim of this thesis was to create a flexible, user-friendly, and reliable method for the simultaneous DNA analysis of up to one thousand DNA variations. To obtain this, we aimed to identify analysis conditions that ensured a low drop-out ratio of the DNA variations under analysis and at the same time guarantee high quality parameters. Another objective was to study different DNA extraction methods from venous blood and saliva. In this case, comparative DNA analysis and APEX-2 genotyping were performed. Saliva DNA analysis is a non-invasive procedure and has many advantages for diagnostic testing compared to DNA extraction from blood. The practical outcome of the current thesis can be applied in molecular diagnostic testing, scientific replication studies, and in genealogical research.

Author

• Krjutškov, Kaarel

Identifier

hdl:10062/14716

OAI identifier oai:identifier

oai:dspace.ut.ee:10062/14716