Subtractive hybridization with covalently modified oligonucleotides

Abstract

The ability to compare two different nucleic acid libraries has permitted inquiries into the role of differentially expressed genes or deleted/inserted genomic sequences involved in the mechanisms of neoplastic transformation, developmental regulation, physiological processing, pathological disorder, and therapeutic efficacy. Subtractive hybridization between two complementary DNA (cDNA) libraries is a powerful tool for identifying differentially expressed genes. In principle, an excess amount of modified subtracter cDNAs derived from cells of a control group are used to bind with tester messenger RNAs (mRNAs) or cDNAs isolated from the cells of interest. Because the subtracter cDNAs are modified to interfere with the amplification processes of reverse transcription (RT) and/or polymerase chain reaction (PCR), all subtracter-bound tester sequences are degraded and only the differentially expressed genes in the tester can be preserved for RT-PCR amplification. To improve the efficiency of subtractive hybridization, we have developed a chemical modification procedure to generate covalently binding cDNAs as the subtracter to capture the homologous tester sequences. We have also proved that the covalently bound duplex hybrids cannot be separated in PCR and thus are removed from the amplified differential gene sequences. Using the novel principle of covalently hybridized subtraction (CHS), we provide an easy, fast, and effective subtractive hybridization method for understanding the alterations of gene expression and/or chromosomal rearrangement in disordered cells in comparison with normal ones, which may reveal targets for gene therapy, eugenic improvement, pharmaceutical drug design, and investigation of etiological mechanisms.