Normal/disease-paired cDNA library subtraction for molecular marker development

This study consists of a novel strategy for the identification of potential cancer exclusively expressed genes, which might lead to the development of valuable diagnostic molecular markers. Normal- and cancer-paired tissues from the same patient were collected and subjected to the construction of primary complementary deoxyribonucleic acid (cDNA) libraries. The cancer cDNA library was generated by subtracting normal cDNAs from the primary cancer library. The remaining clones in the subtracted cancer library were sequenced and Basic Local Alignment Search Tool (BLAST)-analyzed against current nonredundant and est_human databases in the GenBank. The clones that had no matches with any known gene sequences except the human genomic sequences were identified as ideal candidates for potential molecular marker development. The candidate marker sequence and associated primer sequences were identified on the target clone sequence of the region with the least, or no, matched homologs. The specificity of the markers was measured by a polymerase chain reaction test experiment with the DNA templates from different human normal tissues, genomic DNA, and additional patients' tissues. Results from bioinformatical and experimental approaches used suggest that the methodology has a high potential to identify cancer exclusive transcripts. Thus, it might result in the development of diagnostic markers.