Assay and kit for analyzing gene expression
First Claim
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.
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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.
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Citations
39 Claims
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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.
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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. - View Dependent Claims (3, 8, 16, 28, 29, 30, 31, 32, 33, 34)
- overhang, and
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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.
- 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.
Specification