CEBR1-02
Development and validation of molecular diagnostic kits using the nano- and recombinant DNA technology for: (a) Mutation detection & (b) Interferon monitoring
Researchers:
Dr. Salman A. H. Alrokayan
Prof. Khalid M. Abu-Salah,
Dr. Fahad Almajhdi
Dr.Khalid S. Abu Khabar
ABSTRACT:
Mutation Detection
Silicon nanoparticles (1-nm) which are highly efficient blue emitters (three to four fold larger than fluorecein) will be used in this project as biosensors for detecting single-point or multiple mutations using DNA- based modification of these nanoparticles. The approach to be employed in this research project depends on the selective binding of oligonucleotide molecules (synthesized single-stranded mutant type) with amino groups on the surface of silicon nanoparticle. Upon target binding (sample DNA to be tested) silicon fluorescence changes. The change in the type and intensity of fluorescence signal is highly specific and sensitive to the target mutant DNA. The other part of the project will be devoted towards establishing long lasting, reproducible, trasfected cell line for measurement of interferon (IFN) in biological samples.
Interferon Monitoring Kit
Interferon (IFN) is an important protein in innate immunity against many types of viruses. Based on its antiviral action and anti-proliferative activity, it constitutes significant therapeutic molecule in a number of viral diseases and cancers. The bioactivity of IFN, necessary for evaluation of therapeutic IFNs and for diagnostic purposes, is assayed customarily by a viral cytopathic effect assay or by other assays, and these assays require multiple steps such as cell destruction or dye incorporation. The overall objective of this proposal is to develop a newly modified, simple, and high-throughput amenable bioassay for IFN in form of stable genetically engineered cell line clone responsive and selective to IFN. Specifically, we seek to establish stably-transfected cell lines for measurement of IFN in pharmaceutical and biological samples. The aims of the proposal are: (a) microarray profiling and bioinformatics assessment for searching of strong and specific IFN-stimulated promoter, (b) generation of enhanced green fluorescent protein (EGFP) reporter constructs fused with candidate IFN-stimulated promoters (b) evaluation of the potency and selectivity for inducibility by IFN, and (c) generation and evaluation of a cell line that stably express the chosen IFN-inducible promoter-EGFP reporter gene. The assay will be evaluated for use not only with pharmaceutically-prepared IFN formulations but for measurement of IFN-a in biological samples. In contrast to virus inhibition assay, this assay is simple and convenient and requires no virus or cell lysis. Additionally, unlike other reporter assays which require secondary assays, the new approach offers rapid and reproducible system for IFN quantification. This proposal is in response to announced activity of King Saud Univeristy Center of Excellence in Biotechnology Research devoted to develop technologies from basic science that of are of commercial potential.
OBJECTIVES:
- Employment of nanobiotechnology concepts, vis the newly emerging characteristics of silicon nanoparticles in the development of a sensitive or ultra-sensitive diagnostic laboratory methods for detection of single and multiple point mutations such as those of Werding Hoffman disease and Duchene muscular dystrophy.
- Developing the newly discovered methods, after their validation, in the form of kits which are easy to use and easy to commercialize.
- Employment of recombinant DNA concepts and technology in the development of a method aiming at developing a new, simple, and high-throughput amenable bioassay by establishing a stably-transfected cell lines for measurement of INF in pharmaceutical and biological samples such as HBV or HCV-infected patients treated with recombinant human INF-a.
- Translating scientific discoveries into goods and services as rapid and accurate molecular diagnosis increases the chances of survival, prevents resources from being wasted on inappropriate treatment, and helps contain disease.