Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1996-04-04
2002-10-01
Marschel, Ardin H. (Department: 1631)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S501000, C536S023100, C536S024200, C536S024300, C536S024310, C536S024320, C536S025300
Reexamination Certificate
active
06458530
ABSTRACT:
COPYRIGHT NOTICE
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
COMPUTER PROGRAM LISTING APPENDIX
The present specification includes a compact disc labeled “Copy 1” comprising a Computer Program Listing Appendix and an exact duplicate compact disc labeled “Copy 2.” Said Computer Program Listing Appendix was created on May 23, 2002 and comprises text listings of the computer programs written in the “C” language “tags.ccp.txt” (11,505 bytes) and “tags895.ccp.txt” (10,895 bytes). The content of the Computer Program Listing Appendix is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
This invention provides sets of nucleic acid tags, arrays of oligonucleotide probes, nucleic acid-tagged sets of recombinant cells and other compositions, and methods of selecting oligonucleotide probe arrays. The invention relates to the selection and interaction of nucleic acids, and nucleic acids immobilized on solid substrates, including related chemistry, biology, and medical diagnostic uses.
BACKGROUND OF THE INVENTION
Methods of forming large arrays of oligonucleotides and other polymers on a solid substrate are known. Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070), McGall et al., U.S. Pat. No. 5,412,087, Chee et al. SN PCT/US94/12305, and Fodor et al., PCT Publication No. WO 92/10092 describe methods of forming arrays of oligonucleotides and other polymers using, for example, light-directed synthesis techniques.
In the Fodor et al. publication, methods are described for using computer-controlled systems to direct polymer array synthesis. Using the Fodor approach, one heterogenous array of polymers is converted, through simultaneous coupling at multiple reaction sites, into a different heterogenous array. See also, Fodor et al. (1991)
Science,
251: 767-777; Lipshutz et al. (1995)
BioTechniques
19(3): 442-447; Fodor et al. (1993)
Nature
364: 555-556; and Medlin (1995)
Environmental Health Perspectives
244-246. The arrays are typically placed on a solid surface with an area less than 1 inch
2
, although much larger surfaces are optionally used.
Additional methods applicable to polymer synthesis on a substrate are described, e.g., in U.S. Pat. No. 5,384,261, incorporated herein by reference for all purposes. In the methods disclosed in these applications, reagents are delivered to the substrate by flowing or spotting polymer synthesis reagents on predefined regions of the solid substrate. In each instance, certain activated regions of the substrate are physically separated from other regions when the monomer solutions are delivered to the various reaction sites, e.g., by means of groves, wells and the like.
Procedures for synthesizing polymer arrays are referred to herein as very large scale immobilized polymer synthesis (VLSIPS™) procedures. Oligonucleotide VLSIPS™ arrays are useful, for instance, in a variety of procedures for monitoring test nucleic acids in a sample. In probe arrays with multiple probe sets, many distinct hybridization interactions can be monitored simultaneously. However, unwanted hybridization between probes, or between probes and other nucleic acids, can make analysis of multiple hybridizations problematic. This invention solves these and other problems.
SUMMARY OF THE INVENTION
With this invention it is now possible to label and detect many individual components present, inter alia, in molecular, cellular and viral libraries using a limited number of hybridization conditions. Components are labeled with specially selected nucleic acid tags, and the presence of individual tags is monitored by hybridization to a probe array (typically a VLSIPS™ array of oligonucleotide probes). Thus, the tag nucleic acids are labels for the individual components, and the probe array provides a label reader which permits simultaneous detection of a very large number of tag nucleic acids. This facilitates massive parallel analysis of all of the components in a mixture in a single assay.
For instance, as explained herein, all of the members of a cellular library can be tested for response to an environmental stimulus using a mixture of all of the members of the cellular library in a single assay. This is accomplished, e.g., by labeling each member of the cellular library, e.g., by cloning a nucleic acid tag into each cell type in the library, mixing each cell type in the library in an appropriate solution, and exposing part of the solution to the selected environmental stimulus. The distribution of nucleic acids in the library before and after the environmental stimulus is compared by hybridization of the nucleic acids to a VLSIPS™ array, allowing for detection of cells which are specifically affected by the environmental stimulus.
Accordingly, the present invention provides, inter alia, tag nucleic acids, sets of tag nucleic acids, methods of selecting tag nucleic acids, libraries of cells, viruses or the like containing tag nucleic acids, arrays of oligonucleotide probes, arrays of VLSIPS™ probes, methods of selecting arrays of oligonucleotide probes, methods of detecting tag nucleic acids with VLSIPS™ arrays and other features which will become clear upon further reading.
In one class of embodiments, the invention provides a method of selecting a set of tag nucleic acids designed for minimal cross hybridization to a VLSIPS™ array. The absence of cross hybridization facilitates analysis of hybridization patterns to VLSIPS™ arrays, because it reduces ambiguities in the interpretation of hybridization results which arise due to multiple nucleic acid species binding to a single species of probe on the VLSIPS™ array. Thus, in the selection methods of the invention, potential tags are excluded from set of tags where they bind to the same nucleic acid as selected tags under stringent conditions. The selection methods typically include the steps of selecting a specific thermal binding stability for the tag acids against complementary probes, and excluding tags which contain self-complementary regions. Often, the thermal binding stability of the tags is selected by specifying parameters which influence binding stability, such as the length and base composition (e.g., by selecting tags with the same AT to GC ratio of nucleotides) for the tag nucleic acids. In this regard, tags which form more GC bonds upon binding a complementary probe require fewer overall bases to have the same binding stability with a complementary probe as tags which have fewer GC residues. Binding stability is also affected by base stacking interactions, the formation of secondary structures and the choice of solvent in which a tag is bound to a probe.
The size of the tags can vary substantially, but is typically from about 8-150 nucleotides, more typically between 10 and 100 nucleotides, often between about 15 and 30 nucleotides, generally between about 15 and 25 nucleotides and, in one preferred embodiment, about 20 nucleotides in length. In a few applications, the tags are substantially longer than the probes to which they hybridize. The use of longer tags increases the number of tags from which non-cross hybridizing probes can be selected.
The tag nucleic acids are optionally selected to have constant and variable regions, which facilitates elimination of secondary structure arising from self-complementarity, and provides structural features for cloning and amplifying the tags. For instance, PCR binding sites or restriction enzyme sites are optionally incorporated into constant regions in the tags. In other embodiments, short constant regions are added in coding theory methods to prevent misalignment of the tags. Constant regions are optionally cleaved from the tag during processing steps, for instance by cleaving the tag nucleic acids with clas
Davis Ronald W.
Mittmann Michael P.
Morris Macdonald S.
Shoemaker Daniel D.
Affymetrix Inc.
Marschel Ardin H.
Morgan & Lewis & Bockius, LLP
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