Methods for negative selections under solid supports

a technology of solid supports and negative selections, applied in the field of quantitative and isolation, can solve the problems of slow and expensive sequencing, limited genomic sequencing, and laborious process of positional gene cloning,

Inactive Publication Date: 2003-03-13
DELTAGEN PROTEOMICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

0022] The invention described herein provides methods and compositions for the detection and isolation of specific target nucleic acids from a complex mixture of nucleic acids. The methods of this invention enable quantitative comparisons of numerous individual sequences and recovery of those that have specific relative abundance with reference to other sequences in a mixture of nucleic acids, and/or to the same target nucleic acid in a different complex mixture. ...

Problems solved by technology

In general, however, the process of positional gene cloning, i.e., cloning a gene based on its genetic location, is laborious.
However, sequencing, as a systematic approach for genomic analysis, is slow and expensive.
Indeed, genomic sequencing has been limited to a few particularly interesting genes or genetic intervals.
Furthermore, the availability of sample mRNA/cDNA/genomic DNA may be rather limited.
Additionally, the level of each specific nucleic acid molecule (mRNA, cDNA, genomic DNA fragment) must be determined separately with a corresponding specific probe, which may be labor- and resource-intensive.
However, each of these methods has problems, especially when it is an objective to analyze large numbers of targets and the available amounts of sample nucleic acids are a limiting factor.
However, this type of assay is not suited for analysis of large numbers of probes.
The major drawback with this approach is its lack of sensitivity.
It is typically impossible to identify differentially expressed sequences that are present in amounts of less than one (1) occurrence in as much as 1,000 to 10,000 sequences.
In addition, for detection there must be a relative large disparity in expression of a particular sequence.
However, differential hybridization is technically very difficult.
Furthermore, it lacks sensitivity, and is only suited for identification of differentially expressed sequences that are present in relative amounts higher than about o...

Method used

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  • Methods for negative selections under solid supports
  • Methods for negative selections under solid supports
  • Methods for negative selections under solid supports

Examples

Experimental program
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first embodiment

[0091] In a first embodiment, glass beads are treated with 3-glycidoxypropyltrimethoxysilane to generate a terminal epoxide conjugated via a linker to Si atoms on the glass. In a second step, the epoxide is opened with either water or a diol to generate alcohols. Maskos and Southern, 1992, Nucleic Acids Research 20:1679-1684. The resulting siloxane linkage is relatively stable to base hydrolysis. Glass beads are a necessary starting material to produce hydroxyl groups suitable to begin cycles of phosphoramidite chemistry in a conventional automated DNA synthesizer. In some preferred applications, commercially available controlled-pore glass (CPG) or polystyrene supports are employed as beads. Such supports are available with base labile linkers and initial nucleosides attached, by, e.g., Applied Biosystems (Foster City, Calif.). Alternatively, non-porous glass beads, e.g., Ballotini spheres are employed (Maskos and Southern, 1992, Nucleic Acids Research 20:1679-1684).

second embodiment

[0092] In a second embodiment, the linkage is created by the reaction of primary amines with phosphoramidite nucleotides to produce a base-stable linkage. Pon et al., 1988, Biotechniques 6:768-775. In the first step of the reaction an N-P linkage is formed due to nucleophilic attack by nitrogen on phosphorus. This linkage is oxidized in a subsequent step to the phosphoramidate, a stable chemical linkage. Beads that are functionalized with surface primary amines can be obtained from commercial sources.

third embodiment

[0093] In a third embodiment, the capture oligonucleotides are attached to the bead via a phosphodiester bond generated by standard phosphoramidite synthesis utilizing the attack of bead-linked hydroxyl oxygens on the nucleotide phosphorus to produce a phosphodiester bond, following oxidation with molecular iodine. Others have utilized this reaction to generate stable linkages (e.g., Needels et al, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:10700-10704). The key step is the derivatization of appropriate beads such that they contain significant numbers of hydroxyl functional groups on their surface. It is possible to purchase such functionalized beads from a variety of commercial sources; the capture oligonucleotides may be synthesized chemically on the surface of these functionalized beads.

[0094] Generally, standard synthesis chemistries are used, such as phosphoramidite chemistry, as disclosed in Beaucage and Iyer, 1992, Tetrahedron 48:2223-2311, Molko et al., U.S. Pat. No. 4,980,460; ...

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Abstract

The present invention relates to a method for performing negative selections-i.e., identifying agents that can eliminate a cell from a population. More specifically, the invention relates to a method using oligonucleotides covalently linked to a solid support, such as beads, to isolate specific labeled nucleic acid sequences that encode agents which kill or arrest growth in a cell.

Description

[0001] This application is a continuation-in-part of U.S. Ser. No. 08 / 764,191, "Methods for measuring relative amounts of nucleic acids in a complex mixture and retrieval of specific sequences therefrom," WO98 / 26098 (same title), and [VEN002-01 US NATIONAL PHASE] (same title), each of which is expressly incorporated herein in its entirety.I. FIELD OF THE INVENTION[0002] The present invention relates generally to methods and compositions for the quantitation and isolation of specific nucleic acids from complex mixtures of nucleic acids. The methods of the invention allow for the comparative assessment of the expression levels of genes in samples derived from different sources, e.g., different tissue or cell types, disease- or development stages. The invention also relates to sorting large populations of nucleic acids based on quantitative measures of abundance in such a manner that the nucleic acids can be retrieved for subsequent molecular biological experiments. The invention has u...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12N15/1065C12N15/1075C12Q1/6809C12Q2545/114C12Q2563/149C12Q2565/514C12Q2565/515
Inventor KAMB, C. ALEXANDER
Owner DELTAGEN PROTEOMICS
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