Method of generating nucleic acid oligomers of known composition

#This invention pertains to a method for providing oligomers of known subunit sequence which hybridize specifically to DNA and RNA having complementary nucleotide sequences, in which the desired oligomers are released from selected storage sites on a substrate by locally denaturing double-stranded complexes at the storage sites containing the desired oligomers. The released oligomers are oligonucleotides or oligonucleotide analogs, and are useful in schemes for determining solutions to mathematical problems, in methods wherein hybridizing oligomers are used to encrypt and transmit data, in diagnostic and screening assay methodologies, and as primers or building blocks for synthesizing larger polynucleotides. The present invention also features providing oligomers having desired subunit sequences from a device comprising a substrate supporting an array of oligomer-storing depot sites made by a novel method for the synthesis of DNA arrays which utilizes local melting of hybridized DNA and produces a set of substrate-attached oligomers of known subunit sequence. The present invention has applications in the fields of molecular computation, biochemistry, molecular biology, pharmacology, medical diagnostic technology, and data encryption and transmission.

#All publications and patent applications herein are incorporated by reference, fully as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

#Various strategies for finding solutions to mathematical problems have been devised which use sets of DNA oligonucleotides having selected length and sequence properties. For example, DNA-based methods are developed for solving a Hamiltonian path problem (Adleman, Science, 1994, Vol. 266, pages 1021-3), a "satisfaction" problem (Lipton, Science, 1995, Vol. 268, pages 542-5), and for performing addition (Guarnieri et al., 1996, Science, vol. 273, pages 220-223) and matrix multiplication (Oliver, J. Molecular Evolution, 1997, Vol. 45, pages 161-7 ) of non-negative numbers. Each computation requires a set of oligonucleotides having properties tailored to the problem to be solved. Thus, a rapid and efficient method for providing custom sets of oligonucleotides having selected sequence and length properties is essential for efficient application of DNA-based computation methods.

#FIG. 1 schematically represents an oligomer-storing device comprising a substrate (1) supporting an array of oligomer depot sites (2). The dotted line indicates the section giving the view shown in FIG. 2.

#FIG. 2 schematically represents a cross-section through a row of depot sites of the oligomer-storing device shown in FIG. 1, for which the substrate (1) is a transparent substrate. A preferred embodiment of the present invention is shown wherein localized heating of a selected depot (3) is achieved by using a source of radiant energy (4) to irradiate the selected depot site through the transparent substrate (1) to release the desired single-stranded oligomers (5).

#FIG. 3 schematically illustrates the seven basic steps of the disclosed ligation/restriction-based method for synthesizing an oligonucleotide array in which .epsilon. strands are hybridized to .gamma. strands in step 3. The steps are described in detail below.

#This invention features methods wherein custom sets of oligonucleotides and/or oligonucleotide analogs having selected subunit sequences are rapidly and efficiently provided by their controlled release from depot sites of an oligomer storage device.

#The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, recombinant DNA, and medical diagnostic technology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; J. M. Polak and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice; Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and, D. M. J. Lilley and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these general texts are herein incorporated by reference.

#Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described.