Collections of uniquely tagged molecules

Details Collections of uniquely tagged molecules Collections of uniquely tagged molecules

1997-10-06

2003-08-19

Horlick, Kenneth R. (Department: 1637)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100

Reexamination Certificate

active

06607878

ABSTRACT:

BACKGROUND AND SUMMARY OF THE INVENTION
This invention is directed to methods and kits for creating and analyzing molecules using uniquely identifiable tags. The invention is also directed to methods and kits that use uniquely identifiable tags for sequencing DNA, for determining mutations, including substitutions, deletions, and additions, in sample genes, and monitoring mRNA populations.
Biologists and chemists have long sought methods to identify a given molecule in a collection of thousands or millions or more of different molecular species. In large mixtures of many different molecules, it is challenging to identify any one molecule or molecular species rapidly. It is often even more difficult to identify several hundred or thousand non-identical or dissimilar species within a collection of many thousands or millions or more of different molecular species. It would be beneficial to functionally tag or “bar code” large numbers of molecular species for rapid, simultaneous identification.
To this end, the idea of using molecules to identify other molecules has emerged. As one example, it is now possible to use combinatorial synthesis techniques to develop large or extremely large collections of different but similar molecular species.
Combinatorial chemistry methods permit the synthesis of large numbers of different molecules in a mixture. In standard “pool and split” combinatorial methods, each molecule in the mixture is associated with a tag or series of tags helpful in determining the identity of the molecule to which the tag is attached. See, for example, Ohlmeyer, M. H. J., et al., “Complex Synthetic Chemical Libraries Indexed With Molecular Tags” Proc. Natl. Acad. Sci. 90:10922-10926, 1993; Pinilla, C., et al., “Versatility of Positional Scanning Synthetic Combinational Libraries for the Identification of Individual Compounds” Drug Devel. Res. 33:133-145, 1994; Gallop, M. A., et al. “Applications for Combinational Technologies to Drug Discovery. *1. Background and Peptide Combinational Libraries.”
J. Med. Chem.
37:1233-1251, 1994; Gordon, E. M., et al., “Applications of Combinational Technologies to Drug Discovery. 2. Combinational Organic Synthesis, Library Screening Strategies, and Future Directions.”
J. Med. Chem.
37:1385-1401, 1994; Janda, K. D., “Tagged Versus Untagged Libraries: Methods for the Generation and Screening of Combinational Chemical Libraries.”
Proc. Natl. Acad. Sci.
91:10779-10785, 1994; Dower, W. J., et al., PCT/US92/07815, WO 93/06121 “Method of Synthesizing Diverse Collections of Oligomers”; Matson, R. S. et al., U.S. Pat. No. 5,429,807, “Method and Apparatus for Creating Biopolymer Arrays on a Solid Support Surface”; Southern, E. M., et al., “Arrays of Complementary Oligonucleotides for Analyzing the Hybridization Behavior of Nucleic Acids.”
Nucl. Acids. Res.
22:1368-1373, 1994; Southern, E. M., “DNA Fingerprinting by Hybridization to Oligonucleotide Arrays.” Electrophoresis 16:1539-1542, 1995; Drmanac, R. T. and Crkvenjakov, R. B., “Method of Determining an Ordered Sequence of Subfragments of a Nucleic Acid Fragment by Hybridization of a Oligonucleotide Probes” U.S. Pat. No. 5,492,806; Drmanac, R. T. and Crkvenjakov, R. B., “Method of Sequencing by Hybridization of Oligonucleotide Probes” U.S. Pat. No. 5,525,464; McGall, G. H., et al., “Spatially-Addressable Immobilization of Oligonucleotides and Other Biological Polymers on Surfaces” U.S. Pat. No. 5,412,087; Dower, W. J. and Fodor, S. P. A., “Sequencing of Surface Immobilized Polymers Utilizing Microfluorescence Detection” U.S. Pat. No. 5,547,839; Fodor, S. P. A., et al., “Array of Oligonucleotides on a Solid Substrate” U.S. Pat. No. 5,445,934; and Fodor, S. P. A., “Synthesis and Screening of Immobilized Oligonucleotide Arrays” U.S. Pat. No. 5,510,270. Typically, a combinatorial synthesis will proceed in “stages” with two or more reaction vessels per stage. The purpose of each reaction vessel is to add a unique chemical moiety to a growing collection of chemical compounds.
Each moiety is also associated with a uniquely identifiable “tag.” The tag is typically attached to the same solid support to which the growing chemical compounds are attached. Thus, attachment of a tag to a solid support (typically a bead) conveys the information about the bead concerning the particular reaction vessel through which the bead has passed during the synthesis. In pool and split strategies, after the tags are attached in a particular stage, all of the reaction vessel contents are pooled, mixed, and divided and dispersed into new reaction vessels in the next stage. Each moiety added in each new reaction vessel will also be associated with a unique tag added to the beads. Thus, the collection of tag molecules on each bead conveys the “synthetic pathway” though which the particular bead was placed.
In standard screening of combinatorial chemistry libraries, information regarding the order of addition of the tags and the linkage of tags to one another is not needed. Combinatorial chemical libraries are typically screened in the hopes of finding a few members giving the strongest positive signals in the screening assay. The screens are typically performed in separate reaction wells, where one or a few members of the combinatorial library (one or a few beads) is placed in each well. If a particular member scores positively, the composition of the compound can be determined by looking at the tags that are attached to the bead to which the compound is (or was) attached. If one is examining the tags attached to only a single bead, then the synthetic pathway can be identified.
For example, suppose that in the construction of a particular combinatorial chemical library that there are four parallel chemical steps in each synthetic stage, and that there are four synthetic stages each linked by a pool and split step. If there are 16 uniquely identifiable tag molecules available, then each bead will have four tag molecules associated with it (corresponding to the four stages of chemical synthesis). Each tag molecule becomes a marker for each of the 16 reaction vessels. Any particular bead will have traveled through four of the reaction vessels during the procedure, and the four tag molecules that become associated with the bead will reveal the “synthetic pathway” of the bead provided that each bead is examined separately.
There are instances, however, in which it would be desirable to examine 100 positive beads together. If each bead contains four types of tag molecules and all of the tags are released from the beads and examined together, it will not be possible to determine the 100 different pathways that were used. Since there are only 16 different tag types, many pathways will use the same tag types in some but not all of their synthetic steps.
Thus, a primary difficulty in using such techniques lies in screening all of the species for those containing the desired activities or properties and then analyzing the molecular makeup of such species. To this end it has been proposed to use unique combinations of nucleotides to identify protein sequences that are constructed with combinatorial synthesis techniques. Brenner, S. & R. A. Lerner, “Encoded Combinatorial Chemistry,”
Proc. Natl. Acad. Sci
. USA 89:5381-83 (June 1992). The Brenner method decodes the unique combinations of nucleotides by actually sequencing the nucleotide tags. Although this method may permit one to determine the identity of a large number of molecules in a combinatorial library, the method still requires the physical separation of the linked tags (oligonucleotides) themselves for individual analysis (by PCR and cloning followed by DNA sequencing). Thus, the method fails to identify a large subset of molecules simultaneously. It merely shifts the need from physical separation and isolation of the beads to physical separation and isolation (cloning) of amplicons. In addition, the Brenner method would not permit the use of tags as a substitute for traditional DNA sequencing methods, since the analysis of the tags relies on traditional DNA sequenci

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