Preparation of nucleic acid samples

Abstract

The presently claimed invention provides methods, compositions, and apparatus for studying nucleic acids. Specifically, the present invention provides a novel enrichment and labeling strategy for ribonucleic acids. In one embodiment, the invention provides enriching for a population of interest in a complex population by diminishing the presence of a target sequence. In a further embodiment, the invention can be used to reproducibly label and detect extremely small amounts of nucleic acids.

1 . A method of preparing a nucleic acid comprising: 
 increasing the relative percentage of a population of nucleic acids of interest within a mixed population of nucleic acids, wherein said population of interest comprises a plurality of nucleic acid sequences, comprising: 
 (a) contacting a nucleic acid sample with a bait molecule, wherein said bait molecule is capable of complexing specifically to a target sequence, but not to said sequences in said population of interest, under such conditions as to allow for the formation of a bait:target complex;  
 (b) removing said bait:target complex from said mixed population thereby resulting in an increase in the relative percentage of said population of interest;  
   fragmenting the sequences from said population of interest to produce fragments; and    adding a signal moiety to the fragments.    
     
     
         2 . The method of  claim 1  wherein the nucleic acid sample is an RNA sample.  
     
     
         3 . The method of  claim 1  wherein the nucleic acid sample is derived from a prokaryotic organism.  
     
     
         4 . The method of  claim 1  wherein the nucleic acid sample is derived from a gram negative prokaryotic organism.  
     
     
         5 . The method of  claim 1  wherein the nucleic acid sample is derived from  E. coli.    
     
     
         6 . The method of  claim 1  wherein said population of interest is messenger RNA (mRNA.)  
     
     
         7 . The method of  claim 1  wherein said target sequence is stable RNA.  
     
     
         8 . The method of  claim 1  wherein said target sequence is ribosomal RNA (rRNA).  
     
     
         9 . The method of  claim 1  wherein said target sequence is 23S RNA.  
     
     
         10 . The method of  claim 1  wherein said target sequence is 16S RNA.  
     
     
         11 . The method of  claim 1  wherein said bait molecule is generated exogenously.  
     
     
         12 . The method of  claim 1  wherein said bait molecule is chemically synthesized.  
     
     
         13 . The method of  claim 1  wherein said bait molecule is cloned from single stranded phage DNA.  
     
     
         14 . The method of  claim 1  wherein said bait molecule is synthesized by reverse transcriptase using said target sequence as a template.  
     
     
         15 . The method of  claim 1  wherein the nucleic acid sample is an RNA sample, the bait molecule is DNA, and the bait:target complex is a DNA:RNA hybrid.  
     
     
         16 . The method of  claim 14  wherein said bait molecules are synthesized by reverse transcriptase after the addition of primers comprising at least one of the following sequences:  
       
         
           
                 
                 
                 
               
                     
                     
                 
                     
                   5′-CCTACGGTTACCTTGTT-3′ 
                     
                 
                     
                     
                 
                     
                   5′-TTAACCTTGCGGCCGTACTC-3′ 
                 
                     
                     
                 
                     
                   5′-TCGATTAACGCTTGCACCC-3′ 
                 
                     
                     
                 
                     
                   5′-CCTCACGGTTCATTAGT-3′ 
                 
                     
                     
                 
                     
                   5′-CCATTATACAAAAGGTAC-3′ 
                 
                     
                     
                 
                     
                   5′-CTATAGTAAAGGTTCACGGG-3′ 
                 
                     
                     
                 
                     
                   5′-TCGTCATCACGCCTCAGCCT-3′ 
                 
                     
                     
                 
                     
                   5′-TCCCACATCGTTTCCCAC-3′. 
                 
                     
                     
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         17 . The method of  claim 1  wherein said bait is attached to a solid substrate.  
     
     
         18 . The method of  claim 17  wherein said solid substrate is a bead.  
     
     
         19 . The method of  claim 17  wherein said step of removing said target sequence is accomplished by separating said solid substrate from said mixed population.  
     
     
         20 . The method of  claim 1  wherein said bait is modified to comprise a selectable element.  
     
     
         21 . The method of  claim 20  wherein said selectable element is selected from the group consisting of: a nucleic acid sequence, a ligand, a receptor, an antibody, a haptenic group, an antigen, an enzyme or an enzyme inhibitor.  
     
     
         22 . The method of  claim 20  further comprising the step of exposing said bait:target complex to a reagent capable of binding said selectable element to form a reagent:bait:target complex.  
     
     
         23 . The method of  claim 22  wherein the reagent capable of binding said selectable element is selected from the group consisting of: a nucleic acid sequence, a ligand, a receptor, an antibody, a haptenic group, an antigen, an enzyme or an enzyme inhibitor.  
     
     
         24 . The method of  claim 20  wherein said selectable element is a biotin.  
     
     
         25 . The method of  claim 22  wherein said reagent capable of binding said selectable element is streptavadin.  
     
     
         26 . The method of  claim 22  wherein said step of removing said RNA sequence is accomplished by separating said reagent:bait:target complex from said mixed population.  
     
     
         27 . The method of  claim 26  wherein the reagent:bait:target complex is attached to a solid support.  
     
     
         28 . The method of  claim 15  wherein said step of removing said RNA:DNA hybrid comprises exposing said RNA:DNA hybrid to a reagent which specifically recognizes RNA:DNA hybrids.  
     
     
         29 . The method of  claim 28  wherein said reagent is RNAse H.  
     
     
         30 . The method of  claim 28  wherein said reagent is an antibody.  
     
     
         31 . The method of  claim 1  wherein the step of removing said bait:target complex is a two step process in which the target is removed first and the bait molecule is removed thereafter.  
     
     
         32 . The method of  claim 29  further comprising the step of removing any remaining DNA bait molecules after said target RNA sequence is removed.  
     
     
         33 . The method of  claim 32  wherein said step of removing said DNA bait molecule is accomplished by digestion with DNAse I.  
     
     
         34 . The method of  claim 31  wherein steps (a) and (b) are repeated.  
     
     
         35 . The method of  claim 34  wherein the same bait molecule is used to remove multiple target sequences.  
     
     
         36 . The method of  claim 35  wherein a thermostable RNAse H is used to remove said target sequences from said bait:target complex.  
     
     
         37 . The method of  claim 34  wherein step (a) is performed at a first temperature and step (b) is performed at a second temperature.  
     
     
         38 . The method of  claim 1  wherein said signal moiety is a biotin.  
     
     
         39 . The method of  claim 1  wherein said signal moiety is a PEO-Iodoacetyl Biotin.  
     
     
         40 . The method of  claim 1  wherein the signal moiety is attached to the 5′ ends of said fragments.  
     
     
         41 . The method of  claim 40  wherein after said step of fragmenting, said 5′ ends of said fragments are chemically modified.  
     
     
         42 . The method of  claim 41  wherein the 5′ ends of said fragments are chemically modified by (—S-ATP and T4 kinase.  
     
     
         43 . The method of  claim 40  wherein said chemical modification results in the addition of a thiol group to the 5′ end of said fragments.  
     
     
         44 . The method of  claim 43  wherein said detectable signal moiety is PEO-Iodoacetyl Biotin.  
     
     
         45 . A method of increasing the relative percentage of a nucleic acid population of interest within a mixed population of nucleic acids, wherein said population of interest comprises a plurality of nucleic acid sequences, comprising: 
 (a) contacting a nucleic acid sample with a bait molecule, wherein said bait molecule is capable of hybridizing specifically to a target sequence but not to said sequences in said population of interest, under such conditions as to allow for the formation of a bait:target complex; and    (b) removing said bait:target complex from said mixed population thereby resulting in an increase in the relative percentage of said nucleic acid population of interest.    
     
     
         46 . The method of  claim 45  wherein the nucleic acid sample is an RNA sample.  
     
     
         47 . The method of  claim 45  wherein the nucleic acid sample is derived from a prokaryotic organism.  
     
     
         48 . The method of  claim 45  wherein the nucleic acid sample is derived from a gram negative prokaryotic organism.  
     
     
         49 . The method of  claim 45  wherein the nucleic acid sample is derived from  E. coli.    
     
     
         50 . A compound having the formula: 
 n-S-acetyl-PEO-sig    wherein n is a polynucleotide, S is thiol, acetyl is an acetyl functional group, PEO is polyethelene oxide, and sig is a signal moiety.    
     
     
         51 . The compound of  claim 50  wherein said signal moiety is a biotin.  
     
     
         52 . The compound of  claim 50  wherein said polynucleotide is a DNA.  
     
     
         53 . The compound of  claim 50  wherein said polynucleotide is an RNA.  
     
     
         54 . The compound of  claim 50  wherein said polynucleotide is an mRNA.  
     
     
         55 . The compound of  claim 50  wherein said thiol group is at the 5′ of said polynucleotide.  
     
     
         56 . A method for labeling a polynucleotide comprising: 
 contacting said polynucleotide with PEO-iodoacetyl conjugated to a signal moiety under conditions such that the PEO-iodoacetyl will attach to said polynucleotide.    
     
     
         57 . The method of  claim 56  wherein said polynucleotide comprises a thiol group.  
     
     
         58 . The method of  claim 57  wherein said thiol group is at the 5′ of said polynucleotide.  
     
     
         59 . The method of  claim 58  wherein said signal moiety is a biotin.  
     
     
         60 . The method of  claim 56  wherein said polynucleotide is a DNA.  
     
     
         61 . The method of  claim 56  wherein said polynucleotide is an RNA.  
     
     
         62 . The method of  claim 56  wherein said polynucleotide is an mRNA.  
     
     
         63 . A method for labeling a polynucleotide comprising: 
 contacting said polynucleotide with a reactive thiol group to form a thiolated polynucleotide;    contacting said thiolated polynucleotide with a signal moiety capable of reacting with said thiolated polynucleotide under appropriate conditions such that said signal moiety is attached to said polynucleotide.    
     
     
         64 . The method of  claim 63  wherein said step of creating a thiol group comprises contacting said polynucleotide with a gamma S ATP and a kinase.  
     
     
         65 . The method of  claim 63  wherein said signal moiety is a biotin.  
     
     
         66 . The method of  claim 63  wherein said polynucleotide is a DNA.  
     
     
         67 . The method of  claim 63  wherein said polynucleotide is an RNA.  
     
     
         68 . The method of  claim 63  wherein said polynucleotide is an mRNA.  
     
     
         69 . A method of labeling prokaryotic mRNA comprising: 
 obtaining a population of RNA comprising both stable RNA and mRNA from a prokaryotic organism;    increasing the relative percentage of mRNA in said population of RNA comprising the steps of;    exposing said population of RNA to a plurality of DNA bait molecules which are complementary to at least a portion of the stable RNA in said population of RNA under such conditions as to allow for the formation of DNA:RNA hybrids;    exposing said DNA:RNA hybrids to RNAse H to remove the RNA from said RNA:DNA hybrids, producing a sample comprising of DNA and mRNA; and    exposing said sample comprising of DNA and mRNA to DNAse thus increasing the relative percentage of mRNA within said population of mRNA;    fragmenting said mRNA to form mRNA fragments;    exposing said mRNA fragments to γ-S-ATP and T4 kinase to produce reactive thiol groups at the 5′ ends of said mRNA fragments, thereby forming thiolated mRNA fragments; and    exposing said thiolated mRNA fragments to PEO-Iodoacetyl-Biotin such that a stable thio-ether bond is formed between said thiolated mRNA fragments and said PEO-Iodoacetyl-Biotin.