Assay and kit for analyzing gene expression

Abstract

It is one objective of the present invention to obtain reproducible representations of expressed mRNA molecules by exploiting a novel technique that relies on short, single stranded polynucleotide tags. In one preferred embodiment, only one polynucleotide tag is obtained from each mRNA molecule, and relatively simple counting statistics can thus be applied after identification and sampling of the different tags, or a subset of tags being present in the population of representative tags. The tags according to the present invention are preferably single stranded polynucleotide tags obtained by subjecting genetic material derived from a biological sample to at least one site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands in the process of generating the at least one single stranded polynucleotide tag. Accordingly, the present invention demonstrates that nicking endonucleases may advantageously be used for obtaining and isolating ssDNA tags. This novel approach in one embodiment eliminates the occurrence of any linker sequence in the ssDNA tag, and it eliminates the presence of a complementary strand in the isolated polynucleotide tag. The lack of linker sequence in the tag and the lack of any complementary strand serves to reduce the huge complexities associated with the analysis of expressed molecules in a biological sample.

1 . A kit for performing or assaying expression profiling of polynucleotides by determining the presence or sequence of a single stranded polynucleotide tag, said kit comprising 
 a) at least one site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag,    b) at least one adapter oligonucleotide comprising a recognition motif for said site-specific nicking endonuclease, and    c) at least one identifying linker oligonucleotide for identifying said at least one single stranded polynucleotide tag.    
     
     
         2 . A kit for performing or assaying expression profiling of polynucleotides by determining the presence or sequence of a single stranded polynucleotide tag, said kit comprising 
 a) a first identifying linker oligonucleotide for identifying said at least one single stranded polynucleotide tag, said first identifying linker oligonucleotide comprising a single stranded part forming a 5′ overhang, and    b) a second identifying linker oligonucleotide for identifying said at least one single stranded polynucleotide tag, said second identifying linker oligonucleotide comprising a single stranded part forming a 3′ overhang.    
     
     
         3 . The kit according to  claim 2  and further comprising at least one adapter oligonucleotide comprising at least one recognition motif for a site-specific nicking endonuclease.  
     
     
         4 . A kit for performing or assaying expression profiling of polynucleotides by determining the presence or sequence of a single stranded polynucleotide tag, said kit comprising 
 a) at least one site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag,    b) at least one adapter oligonucleotide comprising a recognition motif for said site-specific nicking endonuclease, and    c) at least one molecular identifier facilitating sorting and/or detection of a single stranded polynucleotide tag attached to an identifying linker oligonucleotide for identifying said at least one single stranded polynucleotide tag,    wherein the molecular identifier is selected from the group consisting of 
 i) an epitope,  
 ii) a molecule comprised of a predetermined number of subunits having essentially the same charge, mass, hydrophobic properties, three dimensional structure, wherein different molecular identifiers each comprise a different number of subunits, and wherein said difference in the number of subunits makes it possible to separate or identify individual molecular identifiers when subjecting these to a separation or identification process,  
 iii) a dsDNA or ssDNA oligonucleotide of predetermined length or sequence,  
 iv) a peptide of predetermined length or sequence,  
 v) a first molecule capable of binding a second molecule,  
 vi) a linarized plasmid,  
 vii) a molecule comprising an electromagnetic property or a paramagnetic property, and  
 viii) a moiety capable of emitting an electromagnetic radiation after excitation.  
   
     
     
         5 . The kit according to  claim 4 , wherein said one or more molecular identifiers are attached to said adapter oligonucleotide.  
     
     
         6 . The kit according to  claim 1 , wherein the site-specific nicking endonuclease is of the N.BstNB I type.  
     
     
         7 . The kit according to  claim 1 , wherein the adapter oligonucleotide further comprises one or more recognition motifs, or part thereof, for one or more site-specific restriction endonucleases.  
     
     
         8 . The kit according to  claim 3 , wherein the adapter oligonucleotide further comprises one or more recognition motifs, or part thereof, for one or more site-specific restriction endonucleases.  
     
     
         9 . The kit according to  claim 4 , wherein the adapter oligonucleotide further comprises one or more recognition motifs, or part thereof, for one or more site-specific restriction endonucleases.  
     
     
         10 . The kit according to  claim 7 , wherein the site-specific restriction endonucleases are selected from the group consisting of site-specific restriction endonucleases of type II and site-specific restriction endonucleases of type IIs.  
     
     
         11 . The kit according to  claim 1 , wherein the recognition motif in the adapter oligonucleotide is recognized by different cleavage agents selected from the group consisting of site-specific nicking endonucleases and site-specific restriction endonucleases.  
     
     
         12 . The kit according to  claim 1 , wherein the adapter is in single stranded form and comprises one nucleotide strand, which, together with the complementary strand, generates said one or more recognition motifs.  
     
     
         13 . The kit according to  claim 1 , wherein the adapter is single stranded and comprises one strand of said one or more recognition motifs.  
     
     
         14 . The kit according to  claim 1 , wherein the adapter is double stranded.  
     
     
         15 . The kit according to  claim 1 , wherein the identifying linker oligonucleotide comprises a double stranded part comprising complementary nucleotide strands and/or at least one single stranded part.  
     
     
         16 . The kit according to  claim 2 , wherein the first and second identifying linker oligonucleotides comprise a double stranded part comprising complementary nucleotide strands and/or at least one single stranded part.  
     
     
         17 . The kit according to  claim 15 , wherein the identifying linker oligonucleotide(s) comprises a double stranded part comprising complementary nucleotide strands and/or at least two single stranded parts.  
     
     
         18 . The kit according to  claim 1 , wherein the identifying linker oligonucleotide(s) is exclusively single stranded.  
     
     
         19 . The kit according to  claim 1 , wherein the identifying linker oligonucleotide(s) is a double stranded linker oligonucleotide having a 3′ or 5′ overhang comprising a predetermined sequence capable of hybridizing under suitable conditions to a single stranded polynucleotide tag comprising a sequence that is complementary to the predetermined sequence of the overhang of the identifying linker oligonucleotide.  
     
     
         20 . The kit according to  claim 1 , wherein the identifying linker oligonucleotide(s) is linked to a solid support.  
     
     
         21 . The kit according to  claim 20 , wherein the solid support includes a hybridization array.  
     
     
         22 . The kit according to  claim 1 , wherein said adapter oligonucleotide comprises one or more of 
 i) a molecular identifier facilitating sorting and/or detection of a single stranded polynucleotide tag attached to the identifying linker oligonucleotide,    ii) a selectively identifiable label, and    iii) a recognition motif for a site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag.    
     
     
         23 . The kit according to  claim 1 , wherein said adapter oligonucleotide comprises a molecular identifier facilitating sorting and/or detection of a single stranded polynucleotide tag attached to the identifying linker oligonucleotide,  
     
     
         24 . The kit according to  claim 1 , wherein said adapter oligonucleotide comprises a selectively identifiable label.  
     
     
         25 . The kit according to  claim 1 , wherein said adapter oligonucleotide comprises a recognition-motif for a site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag.  
     
     
         26 . The kit according to  claim 4 , wherein said adapter oligonucleotide further comprises a selectively identifiable label.  
     
     
         27 . The kit according to  claim 4 , wherein said adapter oligonucleotide further comprises a recognition motif for a site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag.  
     
     
         28 . The kit according to  claim 2 , wherein said first and/or said second identifying linker oligonucleotide(s) comprises one or more of 
 i) a molecular identifier facilitating sorting and/or detection of a single stranded polynucleotide tag attached to the identifying linker oligonucleotide,    ii) a selectively identifiable label, and    ii) a recognition motif for a site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag.    
     
     
         29 . The kit according to  claim 2 , wherein said first and/or said second identifying linker oligonucleotide(s) comprises a molecular identifier facilitating sorting and/or detection of a single stranded polynucleotide tag attached to the identifying linker oligonucleotide.  
     
     
         30 . The kit according to  claim 2 , wherein said first and/or said second identifying linker oligonucleotide(s) comprises a selectively identifiable label.  
     
     
         31 . The kit according to  claim 2 , wherein said first and/or said second identifying linker oligonucleotide(s) comprises a recognition motif for a site-specific nicking endonuclease capable of i) recognizing a predetermined nucleotide motif comprising complementary nucleotide strands and ii) cleaving only one of said complementary strands, said cleavage generating at least one single stranded polynucleotide tag.  
     
     
         32 . The kit according to  claim 2 , wherein said first and/or second identifying linker oligonucleotide(s) comprises one or more molecular identifiers facilitating sorting and/or detection of the single stranded polynucleotide tag attached to said identifying linker oligonucleotide(s), wherein the molecular identifier is selected from the group consisting of 
 i) an epitope,    ii) a molecule comprised of a predetermined number of subunits having essentially the same charge, mass, hydrophobic properties, three dimensional structure, wherein different molecular identifiers each comprise a different number of subunits, and wherein said difference in the number of subunits makes it possible to separate or identify individual molecular identifiers when subjecting these to a separation or identification process,    iii) a dsDNA or ssDNA oligonucleotide of predetermined length or sequence,    iv) a peptide of predetermined length or sequence,    v) a first molecule capable of binding a second molecule,    vi) a linarized plasmid,    vii) a molecule comprising an electromagnetic property or a paramagnetic property, and    viii) a moiety capable of emitting an electromagnetic radiation after excitation.    
     
     
         33 . The kit according to  claim 32 , wherein said one or more molecular identifiers are attached to said first and/or said second identifying linker oligonucleotide(s).  
     
     
         34 . The kit according to  claim 3 , wherein said adapter oligonucleotide comprises one or more molecular identifiers facilitating sorting and/or detection of a single stranded polynucleotide tag attached to an identifying linker oligonucleotide, wherein the molecular identifier is selected from the group consisting of 
 i) an epitope,    ii) a molecule comprised of a predetermined number of subunits having essentially the same charge, mass, hydrophobic properties, three dimensional structure, wherein different molecular identifiers each comprise a different number of subunits, and wherein said difference in the number of subunits makes it possible to separate or identify individual molecular identifiers when subjecting these to a separation or identification process,    iii) a dsDNA or ssDNA oligonucleotide of predetermined length or sequence,    iv) a peptide of predetermined length or sequence,    v) a first molecule capable of binding a second molecule,    vi) a linarized plasmid,    vii) a molecule comprising an electromagnetic property or a paramagnetic property, and    viii) a moiety capable of emitting an electromagnetic radiation after excitation.    
     
     
         35 . A solid support comprising a hybridization array comprising a plurality of ordered first identifying linker oligonucleotides, wherein at least one of said first identifying linker oligonucleotides comprises a single stranded nucleotide sequence hybridized to at least one single stranded polynucleotide tag comprising a sequence complementary thereto.  
     
     
         36 . The solid support according to  claim 35 , wherein the single stranded poly-nucleotide tag is obtained by the method comprising the steps of 
 i) providing at least one ribonucleic acid from the biological sample,    ii) obtaining at least one double stranded polynucleotide comprising two complementary strands by using the at least one ribonucleic acid provided in step i) as a template for the synthesis of a polynucleotide strand complementary to the at least one ribonucleic acid,    iii) providing at least one site-specific restriction endonuclease capable of recognizing a recognition motif comprised in the double stranded polynucleotide comprising complementary strands and cleaving the double stranded polynucleotide obtained in step ii) into at least two fragments,    iv) contacting and cleaving the at least one double stranded polynucleotide obtained in step ii) with the at least one site-specific restriction endonuclease provided in step iii),    v) obtaining at least one double stranded polynucleotide fragment by cleaving the at least one double stranded polynucleotide contacted with the at least one site-specific restriction endonuclease in step iv),    vi) providing at least one adapter oligonucleotide comprising at least one recognition motif for at least one site-specific nicking endonuclease, wherein said motif comprises a double stranded oligonucleotide comprising complementary strands, wherein the adapter is capable of being linked together with the at least one double stranded polynucleotide fragment obtained in step v),    vii) obtaining at least one chimeric polynucleotide by linking together the at least one double stranded polynucleotide fragment obtained in step v) and the at least one adapter oligonucleotide provided in step vi),    viii) providing at least one site-specific nicking endonuclease capable of recognizing a recognition motif comprised in the double stranded chimeric polynucleotide comprising complementary strands and cleaving only one of the complementary strands of the chimeric polynucleotide obtained in step vii),    ix) contacting and cleaving the at least one chimeric polynucleotide obtained in step vii) with the at least one site-specific nicking endonuclease provided in step viii), and    x) obtaining at least one single stranded polynucleotide tag.    
     
     
         37 . The solid support according to  claim 35 , wherein the single stranded poly-nucleotide tag is obtained by the method comprising the steps of 
 i) providing at least one ribonucleic acid from the biological sample,    ii) obtaining at least one double stranded polynucleotide comprising two complementary strands by using the at least one ribonucleic acid provided in step i) as a template for the synthesis of a polynucleotide strand complementary to the at least one ribonucleic acid,    iii) providing at least one site-specific restriction endonuclease capable of recognizing a recognition motif comprised in the double stranded polynucleotide comprising complementary strands and cleaving the double stranded polynucleotide obtained in step ii) into at least two fragments,    iv) contacting and cleaving the at least one double stranded polynucleotide obtained in step ii) with the at least one site-specific restriction endonuclease provided in step iii),    v) obtaining at least one double stranded polynucleotide fragment by cleaving the at least one double stranded polynucleotide contacted with the at least one site-specific restriction endonuclease in step iv),    vi) providing at least one adapter oligonucleotide comprising at least one recognition motif for at least one site-specific nicking endonuclease, wherein said motif comprises a double stranded oligonucleotide comprising complementary strands, wherein the adapter is capable of being linked together with the at least one double stranded polynucleotide fragment obtained in step v),    vii) obtaining at least one double stranded chimeric polynucleotide by linking together the at least one double stranded polynucleotide fragment obtained in step v) and the at least one adapter oligonucleotide provided in step vi),    viii) providing at least one further site-specific restriction endonuclease capable of recognizing a recognition motif comprised in the double stranded chimeric polynucleotide comprising complementary strands and cleaving both of the complementary strands of the chimeric polynucleotide provided in step vii),    ix) contacting and cleaving the at least one chimeric polynucleotide obtained in step vii) with the at least one further site-specific restriction endonuclease provided in step viii),    x) obtaining at least one chimeric polynucleotide fragment by cleaving the at least one chimeric polynucleotide contacted with the at least one further site-specific restriction endonuclease in step ix),    xi) providing at least one site-specific nicking endonuclease capable of recognizing a recognition motif comprised in the double stranded chimeric polynucleotide fragment comprising complementary strands and cleaving only one of the complementary strands of the chimeric polynucleotide fragment obtained in step x),    xii) contacting and cleaving the at least one chimeric polynucleotide fragment obtained in step x) with the at least one site-specific nicking endonuclease provided in step xi), and    xiii) obtaining at least one single stranded polynucleotide tag.    
     
     
         38 . The solid support according to  claim 35 , wherein the single stranded poly-nucleotide tag is obtained by the method comprising the steps of 
 i) providing at least one ribonucleic acid from the biological sample    ii) providing at least one adapter oligonucleotide comprising a part of a recognition motif for at least one site-specific nicking endonuclease, wherein said part comprises a single oligonucleotide strand which, together with a complementary strand, forms a recognition motif for at least one site-specific nicking endonuclease,    iii) obtaining at least one chimeric polynucleotide by linking together the at least one ribonucleic acid of step i) with the at least one adapter oligonucleotide of step ii),    iv) obtaining at least one double stranded chimeric polynucleotide comprising an adapter oligonucleotide part by using the chimeric polynucleotide of step iii) as a template for the synthesis of a polynucleotide strand complementary to said chimeric polynucleotide,    v) providing at least one site-specific restriction endonuclease capable of recognizing a recognition motif comprised in the double stranded polynucleotide comprising complementary strands and cleaving the double stranded polynucleotide obtained in step iv) into at least two fragments,    vi) contacting and cleaving the at least one double stranded chimeric polynucleotide obtained in step    iv) with the at least one site-specific restriction endonuclease provided in step v),    vii) obtaining at least one double stranded chimeric polynucleotide fragment by cleaving the at least one double stranded chimeric polynucleotide contacted with the at least one site-specific restriction endonuclease in step vi),    viii) providing at least one site-specific nicking endonuclease capable of recognizing a recognition motif comprised in the double stranded chimeric polynucleotide fragment comprising complementary strands and cleaving only one of the complementary strands of the chimeric polynucleotide fragment obtained in step vii),    ix) contacting and cleaving the at least one chimeric polynucleotide fragment obtained in step vii) with the at least one site-specific nicking endonuclease provided in step viii), and    x) obtaining at least one single stranded polynucleotide tag.    
     
     
         39 . The solid support according to  claim 35 , wherein the single stranded poly-nucleotide tag is obtained by the method comprising the steps of 
 i) providing at least one ribonucleic acid from the biological sample,    ii) providing at least one adapter oligonucleotide comprising a part of a recognition motif for at least one site-specific nicking endonuclease, wherein said part comprises a single oligonucleotide strand which, together with a complementary strand, forms a recognition motif for at least one site-specific nicking endonuclease,    iii) obtaining at least one chimeric polynucleotide by linking together the at least one ribonucleic acid of step i) with the at least one adapter oligonucleotide of step ii),    iv) obtaining at least one double stranded chimeric polynucleotide comprising an adapter oligonucleotide part by using the chimeric polynucleotide of step iii) as a template for the synthesis of a polynucleotide strand complementary to said chimeric polynucleotide,    v) providing at least one site-specific nicking endonuclease capable of recognizing a recognition motif comprised in the double stranded chimeric polynucleotide comprising complementary strands and cleaving only one of the complementary strands of the chimeric polynucleotide obtained in step iv),    vi) contacting and cleaving the at least one chimeric polynucleotide obtained in step iv) with the at least one site-specific nicking endonuclease provided in step v), and    vii) obtaining at least one single stranded polynucleotide tag.