Construction of In Situ Oligonucleotide Arrays on Plastic
The concept of DNA arrays was first introduced in the early 1980s, by Sir Edwin Southern. Since then, many research institutions and biotechnology companies have investigated the potential use of arrays in fields ranging from genetic diagnostics to forensics investigations. A 64-channel automated chemical delivery system, known as the Southern Array Maker, which synthesizes oligonucleotides directly onto an aminated polypropylene substrate has been constructed. Many different arrays have been synthesized for the purpose of detecting single point mutations, which might be either indicators of, or directly responsible for, many different types of genetic diseases and cancers. These include cystic fibrosis, H-ras, K-ras, and other mutations. In addition to the synthesized arrays, we are also looking into various alternative methods of producing both highand low-density DNA arrays. This chapter is intended to demonstrate the synthesis of oligoarrays by in situ method using standard phosphoramidite chemistry. Phosphoramidate linkage to the aminated polypropylene is quite stable under oligo cleavage and deprotection conditions. Oligonucleotide density is approx 3 pmole or 1012 molecules/mm2.
Key WordsDNA microarrays oligoarrays phosphoramidite chemistry probe density solid-phase synthesis hybridization
- 6.Matson, R. S. and Rampal, J. B. (2003) DNA arrays: past, present, and future. Am. Genomic/Proteomic Technol. April/May Issue, 37–44.Google Scholar
- 12.Koester, H. and Coull, J. M. (1990) Membranes with bound oligonucleotides and peptides. US Patent No. 4,923,901.Google Scholar
- 13.Rampal, J. B. (2000) Covalent attachment of biomolecules to derivatized polypropylene supports. US Patent No. 6,013,789.Google Scholar
- 19.The evaluation and purification of synthetic oligonucleotides (1987) DNA Synthesis User Bulletin by Applied Biosystems no. 13.Google Scholar
- 22.Rampal, J. B. (1999) Hybridization detection by pretreating bound singlestranded probes. US Patent No. 5,985,567.Google Scholar