Development of cancer diagnostic techniques based on

Abstract

Over 500,000 deaths were caused by cancer in 2011 alone. Likewise, cancer resulted in about 13% of all human deaths worldwide, and the rates are rising as human life expectancy increases. As a result, researches on diagnosis of cancer with several examinations such as X-ray, computed tomography (CT) scan, magnetic resonance imaging (MRI), biopsy, and bone marrow aspiration have been investigated. However, these are all labor-intensive, expensive, and complex. Therefore, many efforts have recently been dedicated to transform the conventional cancer diagnosis methods into much simpler, cost effective, and faster systems to detect cancer in an early stage. In this thesis, we focused on the development of effective cancer diagnosis methods using two different approaches.In the first part, we developed a molecular diagnostic tool based on the rolling circle amplification (RCA) reaction to identify multiplex single nucleotide polymorphism (SNP) of the cellular tumor antigen p53 (tp53) gene in the breast cancer. We utilized RCA reaction to allow highly sensitive and specific SNP detection. The PCR amplicons ranging from 100 to 160 bp were prepared by eleven sets of template primers for RCA template. Then, eleven pairs of padlock probes were designed for simultaneous detection of targeting SNP loci. The SNP sites were from exon 1, 6, 11, and intron 1, 3, 10 of tp53 gene. The 3’ end of padlock probe was designed as complementary SNP site depending on wild or mutant genotype. A healthy donor’s genomic DNA was analyzed and the elongated RCA products were detected under gel electrophoresis. The genotype of donor was confirmed to be all wide type for selected SNP site only with 0.5 ng of RCA template. We could successfully identify the genotype of SNP in 3 hour including 1.5 h of ligation and 1.5 h of RCA. Thus, we developed RCA reaction system for simple, rapid, sensitive, and multiplex SNP detection for early diagnosis of cancer.In the second part, we demonstrated the se...