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US 2003/0113737 A1Published application
Assay and kit for analyzing gene expression
AssigneeGenomic Expression ApS
InventorMorten Lorentz Pedersen
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AssigneeGenomic Expression ApS
InventorMorten Lorentz Pedersen
Estimating the term…
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.
Method for obtaining at least one single stranded polynucleotide tag from a biological sample, said method comprising the steps of
Method of any of the previous claims, wherein the site-specific restriction endonuclease capable of recognizing complementary strands of a double stranded polynucleotide recognizes a motif comprising 8 nucleotides, or less than 8 nucleotides, such as 7 nucleotides, or less than 7 nucleotides, such as 6 nucleotides, or less than 6 nucleotides, such as 5 nucleotides, or less than 5 nucleotides, such as 4 nucleotides.
Method of any of the previous claims comprising the further step of i) characterizing, ii) separating and/or iii) identifying and/or iv) determining the amount of the at least one single stranded polynucleotide tag from other single stranded polynucleotides and/or double stranded polynucleotides.
Method of any of the previous claims, wherein at least one cleavage agent is attached to a solid support.
Method of any previous claim, wherein a ligation step is carried out by using a ligase that is attached to a solid support.
A kit for performing or assaying expression profiling and comprising at least one cleavage agent including at least one site-specific nicking endonuclease, at least one adapter oligonucleotide, and at least one identifying linker oligonucleotide.
A kit for performing or assaying expression profiling and comprising a first identifying linker oligonucleotide comprising a single stranded part forming a 5′ overhang, and a second identifying linker oligonucleotide comprising a single stranded part forming a 3′ overhang.
A solid support comprising a hybridization array comprising a plurality of ordered first identifying linker oligonucleotides, or a subset of such 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.
Dependencies are inferred from the claim text.