The present invention provides apparatuses and methods for the synthesis of oligonucleotides and related compounds. In particular, the present invention allows to effectively prepare reagents to be fed into an apparatus for the synthesis of such oligomers.

Compounds useful as nucleic acid probes are disclosed. In some embodiments, the compounds have the following structure (I) or a stereoisomer, tautomer or salt thereof, wherein M, L.sup.1a, L.sup.2, L.sup.8, L.sup.1b, L.sup.3, L.sup.5, L.sup.6, L.sup.7, L.sup.4, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sub.a, R.sub.b, R.sub.c, R.sub.d, m, n, q, and w are as defined herein. Methods associated with preparation and use of such compounds are also provided.

Compounds useful as nucleic acid probes are disclosed. In some embodiments, the compounds have the following structure (I) or a stereoisomer, tautomer or salt thereof, wherein M, L.sup.1a, L.sup.2, L.sup.8, L.sup.1b, L.sup.3, L.sup.5, L.sup.6, L.sup.7, L.sup.4, R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sub.a, R.sub.b, R.sub.c, R.sub.d, m, n, q, and w are as defined herein. Methods associated with preparation and use of such compounds are also provided.

A process for making an oligonucleotide, the process including reacting a oligonucleotide precursor with a solid phase support within a reaction vessel, the reaction vessel being coupled to an actuator and having a resting position and inverting the reaction vessel via the actuator such that the reaction vessel is inverted relative to the resting position, wherein the inversion of the reaction vessel results in stirring of the solid phase support within the reaction vessel.

A process for making an oligonucleotide, the process including reacting a oligonucleotide precursor with a solid phase support within a reaction vessel, the reaction vessel being coupled to an actuator and having a resting position and inverting the reaction vessel via the actuator such that the reaction vessel is inverted relative to the resting position, wherein the inversion of the reaction vessel results in stirring of the solid phase support within the reaction vessel.

The present disclosure provides methods of sequencing polynucleotides and compounds, compositions for sequencing of polynucleotides, and synthesis of such compositions. The chemical compounds include nucleotides and their analogs which possess a sugar moiety comprising a cleavable chemical group capping the 3′-OH group and a base, but without covalently bounded dye. The cleavable chemical group is reactive to form covalent bond(s) with a dye used to confirm the presence of the expected base-pairing. The cleavable chemical group capping the 3′OH group can be removed together with the covalently bounded dye. Furthermore, after the cleavable chemical group is cleaved, the free 3′-OH group can be active in continued elongation. Example chemical compounds according to the present disclosure are shown as Formulas (II) and (V):

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Embodiments for the synthesis of sensitive oligonucleotides as well as insensitive oligonucleotides are provided. Sulfur-based groups are used for the protection of exo-amino groups of nucleobases, phosphate groups and 2′—OH groups, and as cleavable linker for linking oligonucleotides to a support. Oligonucleotide syntheses are achieved under typical conditions using phosphoramidite chemistry with important modifications. To prevent replacing sulfur-based protecting groups by acyl groups via cap-exchange, special capping agents are used. To retain hydrophobic tag to assist RP HPLC purification, special phosphoramidites are used in the last synthetic cycle. With the sulfur-based groups for protection and linking, oligonucleotide deprotection and cleavage are achieved via oxidation followed by beta-elimination under mild conditions. Therefore, besides for insensitive oligonucleotide synthesis, the embodiments of the invention are capable for the synthesis of oligonucleotide analogs containing sensitive functional groups that cannot survive the harsh conditions used in prior art oligonucleotide synthesis technologies.

Embodiments for the synthesis of sensitive oligonucleotides as well as insensitive oligonucleotides are provided. Sulfur-based groups are used for the protection of exo-amino groups of nucleobases, phosphate groups and 2′—OH groups, and as cleavable linker for linking oligonucleotides to a support. Oligonucleotide syntheses are achieved under typical conditions using phosphoramidite chemistry with important modifications. To prevent replacing sulfur-based protecting groups by acyl groups via cap-exchange, special capping agents are used. To retain hydrophobic tag to assist RP HPLC purification, special phosphoramidites are used in the last synthetic cycle. With the sulfur-based groups for protection and linking, oligonucleotide deprotection and cleavage are achieved via oxidation followed by beta-elimination under mild conditions. Therefore, besides for insensitive oligonucleotide synthesis, the embodiments of the invention are capable for the synthesis of oligonucleotide analogs containing sensitive functional groups that cannot survive the harsh conditions used in prior art oligonucleotide synthesis technologies.

The present disclosure relates to N-protected NH-rhodamine dyes and their use in nucleic acid detection. In particular, the disclosure relates to methods of making N-protected NH-rhodamine dyes, and methods of use of N-protected NH-rhodamine dyes (e.g., human identification). Certain dyes provided herein have unique spectral properties that complement those in existing dye sets and can be used to expand the number of reporter dyes that can be included for HID applications and other biological assays. Those fluorescent compounds are useful to label synthetic oligonucleotides. Formula (I).

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The present disclosure relates to N-protected NH-rhodamine dyes and their use in nucleic acid detection. In particular, the disclosure relates to methods of making N-protected NH-rhodamine dyes, and methods of use of N-protected NH-rhodamine dyes (e.g., human identification). Certain dyes provided herein have unique spectral properties that complement those in existing dye sets and can be used to expand the number of reporter dyes that can be included for HID applications and other biological assays. Those fluorescent compounds are useful to label synthetic oligonucleotides. Formula (I).

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