Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-02-06
2004-08-24
Riley, Jezia (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S025300, C536S026600, C536S025330, C536S025340
Reexamination Certificate
active
06780982
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel phosphoramidites, including positive and neutrally charged compounds. The present invention also provides charge tags for attachment to materials including solid supports and nucleic acids, wherein the charge tags increase or decrease the net charge of the material. The present invention further provides methods for separating and characterizing molecules based on the charge differentials between modified and unmodified materials.
BACKGROUND OF THE INVENTION
Methods for the detection and characterization of specific nucleic acid sequences and sequence variations have been used to detect the presence of viral or bacterial nucleic acid sequences indicative of an infection and to detect the presence of variants or alleles of genes associated with disease and cancers. These methods also find application in the identification of sources of nucleic acids, as for forensic analysis or for paternity determinations. Various methods are known to the art that may be used to detect and characterize specific nucleic acid sequences and sequence variants. Nonetheless, with the completion of the nucleic acid sequencing of the human genome, as well as the genomes of numerous other organisms such as pathogenic organisms, the demand for fast, reliable, cost-effective and user-friendly tests for the detection of specific nucleic acid sequences continues to grow. Importantly, these tests must be able to create a detectable signal from samples that contain very few copies of the sequence of interest.
There are a number of techniques that have been developed for characterizing specific nucleic acid sequences. Examples of detection techniques include the “TaqMan” or nick-translation PCR assay described in U.S. Pat. No. 5,210,015 to Gelfand et al. (the disclosure of which is herein incorporated by reference), the assays described in U.S. Pat. Nos. 4,775,619 and 5,118,605 to Urdea (the disclosures of which are herein incorporated by reference), the catalytic hybridization amplification assay described in U.S. Pat. No. 5,403,711 to Walder and Walder (the disclosure of which is herein incorporated by reference), the cycling probe assay described in U.S. Pat. Nos. 4,876,187 and 5,011,769 to Duck et al., the target-catalyzed oligonucleotide modification assay described in U.S. Pat. Nos. 6,110,677 and 6,121,001 to Western et al. (the disclosures of which are herein incorporated by reference), the SNP detection methods of Orchid Bioscience in U.S. Pat. No. 5,952,174 (the disclosure of which is herein incorporated by reference), the methods of U.S. Pat. No. 5,882,867 to Ullman et al. (the disclosure of which is herein incorporated by reference) the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188 to Mullis and Mullis et al. (the disclosures of which are herein incorporated by reference) and the ligase chain reaction (LCR) described in U.S. Pat. Nos. 5,427,930 and 5,494,810 to Birkenmeyer et al. and Barany et al. (the disclosures of which are herein incorporated by reference). The above examples are intended to be illustrative of nucleic acid-based detection assays and do not provide an exhaustive list. Each of these techniques requires a detection step for detecting a reaction product that is indicative of a desired target nucleic acid (e.g., detection of cleavage products, extension products, etc.). While a number of advances have been made in the assay methods and detection instrumentation to improve the sensitively, speed, and cost of detection methods the art is still in need of further improved methods, compositions, and systems to make the assays more sensitive and efficient.
SUMMARY OF THE INVENTION
The present invention relates to novel phosphoramidites, including positive and neutrally charged compounds. The present invention also provides charge tags for attachment to materials including solid supports and nucleic acids, wherein the charge tags increase or decrease the net charge of the material. The present invention further provides methods for separating and characterizing molecules based on the charge differentials between modified and unmodified materials.
For example, the present invention provides a composition comprising a charge tag attached to a nucleic acid molecule (e.g., to a terminal end of a nucleic acid molecule). In some embodiments, the charge tag comprises a phosphate group and a positively charged moiety. In some preferred embodiments, the charge tag further comprises a dye. The present invention is not limited by the position of the individual modular components of the charge tag. For example, in some embodiments, the dye is positioned between the nucleic acid and the positively charged moiety, while in other embodiments, the positively charged moiety is positioned between the nucleic acid and the dye. The present invention is also not limited by the number of each type of component in the charge tag (e.g., the number of dyes, positively charged moieties, etc.). For example, in some embodiments, the charge tag comprises first and second positively charged moieties.
In some embodiments of the present invention, the charge tag has a net positive charge. For example, in some embodiments, the charge tag has a net positive charge of 1, 2, 3, etc. In some embodiments, the charge tag possesses a positive charge only under certain reaction conditions (e.g., pH 6-10).
In some embodiments, the charge tag further comprises one or more nucleotides. In some embodiments, the nucleic acid molecule to which the charge tag is attached contains a sequence that is complementary to a target nucleic acid. In some such embodiments, the one or more nucleotides in the charge are not complementary to the target nucleic acid. In other such embodiments, the nucleic acid comprises a first portion complementary to a target nucleic acid and a second portion that is not complementary to said target nucleic acid, wherein the charge tag is attached to the second portion of the nucleic acid (e.g., to a terminal end of the nucleic acid that is located in the second portion).
In some embodiments of the present invention, the nucleic acid and the charge tag have a combined net neutral charge, wherein the charge tag, in isolation, has a net positive charge. In other embodiments, the nucleic acid and the charge tag have a combined net negative charge, wherein the charge tag has a net positive charge.
The present invention is not limited by the nature of the positively charged moiety of the charge tag. Positively charged moieties include, but are not limited to primary amines, secondary amines, tertiary amines, ammonium groups, positively charged metal groups (e.g., caged ions attached to the charge tag through a linking group), and the like.
In some embodiments, the charge tag further comprises a positively charged phosphoramidite or a neutral phosphoramidite. The present invention is not limited by the nature of the positively charged phosphoramidite or the neutral phosphoramidite. For example, in some embodiments, the charge tags comprise a novel phosphoramidite of the present invention.
For example, the present invention provides a composition comprising a positively charged phosphoramidite. In some embodiments, the positively charged phosphoramidite contains one or more positively charged moieties including, but not limited to, primary amine groups, secondary amine groups, tertiary amine groups, ammonium groups, charged metal ions, and the like. In some embodiments, the phosphoramidite has a net positive charge of one. In some particularly preferred embodiments, the phosphoramidite has the structure:
wherein, X is a reactive phosphate group (e.g., PO
4
) and Y is a protecting group (e.g., dimethoxy trityl [DMT]) and/or a protected group (e.g., DMT-protected hydroxyl group).
The present invention further provides a composition comprising a nucleic acid molecule containing a positively or neutrally charged phosphoramidite. The present invention also provides a composition comprising a charge tag a
Allawi Hatim T.
Lyamichev Victor
Neri Bruce P.
Skrzpczynski Zbigniew
Takova Tsetska
Medlen & Carroll LLP
Riley Jezia
Third Wave Technologies Inc.
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