Oligonucleotide scanning arrays: application to high-throughput screening for effective antisense reagents and the study of nucleic acid interactions.
Oligonudeotide scanning arrays are useful tools in the study of nucleic acid interaction. Such arrays of oligonucleotides, corresponding to a full set of complements of a known sequence, can be readily made in a single series of coupling reactions, adding each nudeotide in the complement of the target sequence in turn. The synthesis is carried out on the surface of a solid substrate such as glass or polypropylene that has been modified to allow nucleotide coupling. A mask is used to apply the DNA synthesis reagents in a defined area and is moved by a fixed step size after each coupling reaction so that consecutive couplings overlap a portion of the previous one. The size of the mask and the displacement at each coupling determine the length of the oligonucleotides. A radiolabeled or fluorescently tagged target sequence is hybridised to a scanning array and its interaction with the complementary oligonudeotides is displayed as a hybridisation signal. It is thus possible to determine the exact sequence and lengths of large numbers of interacting sequences in a single hybridisation experiment. The array image is analysed using a computer program (xvseq) that calculates quantitative measurements of the binding strengths. We have found scanning arrays a useful tool not only to find effective antisense reagents, but also to study RNA folding and the mechanisms of heteroduplex formation. In this article, we discuss the format of these arrays, the technology used to fabricate and to read them, and their applications.
Sohail
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University of Oxford, Department of Biochemistry, England, UK. msohail@bioch.ox.ac.uk
Southern
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EM
D000465:Algorithms; D004867:Equipment Design; D009690:Nucleic Acid Conformation; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D016337:RNA, Catalytic / Q000737:chemistry* / Q000235:genetics; D017421:Sequence Analysis / Q000295:instrumentation / Q000379:methods; D013873:Thionucleotides / Q000737:chemistry* / Q000235:genetics
D009692:Nucleic Acid Heteroduplexes; D016376:Oligonucleotides, Antisense; D016337:RNA, Catalytic; D013873:Thionucleotides; D012313:RNA