| Summary: | 碩士 === 朝陽科技大學 === 資訊管理系碩士班 === 94 === In this thesis, we use multiplex polymerase chain reaction (PCR) to locate the functional domain of a cDNA sequence. In order to speed up the experimental procedure and increase the efficiency for generating PCR products, multiple forward primers with the same 3''-UTR reverse primer were designed using meta-heuristic algorithm that we proposed. It is useful in extracting various truncated mutants for quickly locating its functional domain. Several factors, including melting temperature (Tm), primer length, GC contents, internal self-complement, cross-dimmerization, termini limitation, and specificity, were used as criteria for primers design. The goal is to obtain a near-optimal solution with all the primers can be placed in as less tubes as possible for one PCR experiment. To test the efficacy of the proposed method, Homo sapiens ribosomal protein L5 cDNA was used as a test sample for primers design and multiplex PCR experiment. There are totally 48 forward primers and 1 reverse primer that have been designed for duplicating N-terminal truncated mutants of different lengths for a protein. The primers were further divided into 7 clusters (tubes) for PCR experiment in one batch under the same temperature range (53o-57o). The PCR products were verified using the polyacrylamide gel electrophoresis that the functional domain had been located. The proposed method and its accompanied software tool can also be applied to assist the researcher to design multiplex PCR primers for locating the functional domains of Homo sapiens xylosyltransferase I, bacteriophage T4, and other proteins.
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