Methods for negative selections under solid supports

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.

What is claimed is:  
     
         1 . A method of identifying agents that kill or arrest growth in a cell, comprising: 
 (a) introducing an initial library of putative negative selection agents into a cell population;    (b) propagating the cell population;    (c) re-isolating the library components from the propagated cell population; and    (d) subjecting the initial and re-isolated library components to quantitative comparison of the relative amounts of at least one specific library component.    
     
     
         2 . The method of  claim 1 , further comprising as enrichment step (e), in which the library components from step (c) are subjected to one or more cycles of steps (a) through (c).  
     
     
         3 . The method of  claim 1 , wherein the library of putative negative selection agents is a genetic library.  
     
     
         4 . The method of  claim 3 , wherein the library comprises inserts selected from the group consisting of genomic DNA, cDNA and random sequence DNA.  
     
     
         5 . The method of  claim 3 , wherein the genetic library comprises a plurality of inserts, the inserts comprising one or more sequences which, upon expression in a living cell, are capable of differentially altering the phenotype of the host cell.  
     
     
         6 . The method of  claim 5 , wherein expression of the sequences alters host cell gene expression.  
     
     
         7 . The method of  claim 1 , wherein step (d) further comprises: 
 (d1) differentially labeling nucleic acid samples derived from the initial and propagated libraries to generate a first and second labeled nucleic acid sample;    (d2) generating a target pool comprising said first and second nucleic acid samples,    (d3) contacting said target pool with a plurality of solid supports each having attached thereto multiple capture oligonucleotides of a unique sequence under conditions which promote the formation of perfectly matched duplexes between said capture oligonucleotides and nucleic acid molecule components within said target pool; and    (d4) sorting the solid supports according to the relative amount of said first label and said second label.    
     
     
         8 . The method of  claim 7 , wherein the unique capture oligonucleotides attached to the solid supports have a length of from about 10 to about 100 nucleotides.  
     
     
         9 . The method of  claim 7 , wherein the unique capture oligonucleotides attached to the solid supports comprise a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.  
     
     
         10 . The method of  claim 7 , wherein the target nucleic acid molecules have attached thereto unique oligonucleotide identifier tags, each of said tags comprising a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.  
     
     
         11 . The method of  claim 10 , wherein the capture oligonucleotides attached to said solid supports comprise complements of said unique identifier tags.  
     
     
         12 . The method of  claim 7 , wherein said first and said second label are distinguishable fluorescent labels.  
     
     
         13 . The method of  claim 12 , wherein said fluorescent labels are individually selected from the group consisting of 6FAM, HEX, TET, TAMRA, ROX, JOE, 5-FAM, phycoerythrin and R110.  
     
     
         14 . The method of  claim 12 , wherein one of said fluorescent labels is FITC.  
     
     
         15 . The method of  claim 1 , wherein the cell populations in step (a) differ in phenotype.  
     
     
         16 . The method of  claim 1 , wherein the cell populations in step (a) differ in genotype.  
     
     
         17 . The method of  claim 1 , wherein the cell populations in step (a) comprise cells which differ in cell type, tissue type, physiological state, disease state or developmental stage.  
     
     
         18 . The method of  claim 1 , wherein the cell populations in step (a) comprise cancerous and non-cancerous cells, respectively.  
     
     
         19 . The method of  claim 1 , wherein the cell populations in step (a) comprise cells before and after treatment with an agent, respectively.  
     
     
         20 . The method of  claim 19 , wherein the agent is selected from the group consisting of a naturally occurring growth factor, an immunologic factor, a small molecule compound of interest, a putative therapeutic compound, a therapeutic lead compound, and a growth-arresting substance.  
     
     
         21 . A method of identifying a negative selection agent that causes a member of a cell population to be lost from that population, comprising: 
 (a) providing a cell population with a tagged pre-passage library, each member of said library comprising a sequence identifier tag and DNA encoding a corresponding putative negative selection agent;    (b) passaging said cell population to generate a post-passage cell, subpopulation;    (c) isolating from said post-passage cell subpopulation a corresponding tagged post-passage sublibrary;    (d) comparing sequence identifier tags in said pre-passage library to sequence identifier tags in said post-passage sublibrary to identify an sequence identifier tag of a lost library member; and    (e) identifying a negative selection agent that corresponds with said lost library member sequence identifier tag.    
     
     
         22 . The method of  claim 21  wherein said sequence identifier tag comprises a combination of from about 2 to about 6 sequence units in tandem configuration, each unit consisting of from about 7 to about 15 nucleotides.  
     
     
         23 . The method of  claim 22  wherein said combination consists of 3 sequence units, each unit consisting of 8 nucleotides.  
     
     
         24 . The method of  claim 21 , wherein step (d) further comprises: 
 (d1) for each member of said post-passage sublibrary, separating said sequence identifier tag from said corresponding putative negative selection agent;    (e2) segregating each said separated sequence identifier tag; and    (e3) using each said segregated sequence identifier tag to segregate a corresponding negative selection agent from said sublibrary.    
     
     
         25 . The method of  claim 24 , wherein said step of segregating comprises adhering a fluorochrome to said separated tag to form a fluorescent complex, and isolating said fluorescent complex.  
     
     
         26 . The method of  claim 25 , wherein said step of isolating each said fluorescent complex is performed with a fluorescence-activated sorter.  
     
     
         27 . The method of  claim 25 , wherein said fluorochrome is FITC.  
     
     
         28 . The method of  claim 25 , wherein said fluorochrome is attached to a solid support.  
     
     
         29 . The method of  claim 24 , wherein each said segregated tag is a primer for amplifying each said corresponding negative selection agent.  
     
     
         30 . A method of identifying selective lethality agent that kills members of a first cell population but not of a second cell population, comprising: 
 (f) providing both a first cell population and a second cell population with a tagged pre-passage library, each member of said library comprising DNA encoding an sequence identifier tag and a putative selective lethality agent;    (g) passaging said first and said second cell populations;    (h) collecting a first and a second post-passage cell subpopulation;    (i) isolating from said first and second post-passage cell subpopulations corresponding first and second tagged post-passage sublibraries;    (j) comparing said first pre-passage library to said first post-passage sublibrary to identify a first set of lost library members;    (k) comparing said second pre-passage library to said second post-passage sublibrary to identify a second set of lost library members;    (l) identifying a cell-specific lost library member that is in said first set of lost library members but not in said second set of lost library members; and    (m) correlating said cell-specific lost library member with a corresponding selective lethality agent.    
     
     
         31 . The method of  claim 21  or  30 , wherein step (d) further comprises: 
 (d1) transcribing said sequence identifier tags in said pre-passage library and in said post-passage sublibrary;  
 (d2) labeling said pre-passage sequence identifier tags with a first fluorochrome and said post-passage sequence identifier tags with a second fluorochrome;  
 (d3) hybridizing all fluorochrome-labeled sequence identifier tags with a set of support having attached capture oligonucleotides; and  
 (d4) isolating a subpopulation of supports that hybridized only to said first fluorocrome.