HK1154388A - Synthesis and compositions of nucleic acids comprising 2'-terminator nucleotides - Google Patents

Description

Synthesis and compositions of nucleic acids comprising 2' -terminator nucleotides

Technical Field

The present invention relates generally to nucleic acids comprising 2 '-terminator (2' -terminator) nucleotides and methods for their preparation.

Background

Blocked or non-extendable oligonucleotides and polynucleotides include moieties that prevent the addition of additional nucleotides to the oligonucleotides and polynucleotides under a given set of reaction conditions, such as terminator nucleotides. These molecules are commonly used in a variety of nucleic acid technologies. For example, blocked oligonucleotides are used as probes in some applications, such as real-time Polymerase Chain Reaction (PCR), to prevent extension of the probe during the extension step of the reaction. By way of further example, blocked oligonucleotides are also useful as primers in certain applications. For example, Pyrophosphorolysis Activated Polymerization (PAP) is a PCR method comprising primers with a 3' terminal terminator nucleotide that is removed by pyrophosphorolysis prior to primer extension. The PAP method that existed before usually used dideoxy-termination primer and dideoxy-incorporation polymerase. The polymerases used in these pre-existing methods are generally limited in their ability to extend the primer nucleic acid and thus the amplification reaction is inefficient.

There is a need for additional oligonucleotides and polynucleotides comprising terminator nucleotides that can be used in real-time PCR, PAP, and other nucleic acid technologies. The present invention provides oligonucleotides and polynucleotides comprising 2' -terminator nucleotides useful for these uses. These and various other features of the present invention will be apparent upon a complete consideration of the following disclosure.

Summary of The Invention

The present invention relates to oligonucleotides and polynucleotides comprising 2' -terminator nucleotides, which can replace previously existing blocked oligonucleotides and polynucleotides from an economic standpoint, e.g., these blocked oligonucleotides described herein can be readily replaced in various PCR-related methods without sacrificing ease of use. The 2 '-terminator nucleotides of the oligonucleotides and polynucleotides described herein typically have an intact sugar ring or a sugar-like ring (e.g., carbocyclic, etc.), and contain blocking groups (e.g., negatively charged blocking groups, bulky blocking groups, and/or the like) at the 2' -position of these sugar moieties. In addition to methods for the preparation of these oligonucleotides and polynucleotides, the invention also provides related commercial methods and kits.

In one aspect, the invention provides an oligonucleotide or polynucleotide comprising the formula:

wherein Z is O or CH₂(ii) a B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof; BG is a blocking group; r₁H, OH, a hydrophilic group or a hydrophobic group; x is a nucleotide or nucleotide analog; n is an integer greater than 0;represents a single bond or a double bond. In certain embodiments, at least one label (e.g., donor moiety, quencher moiety, acceptor moiety, reporter moiety, etc.) is attached to the oligonucleotide or polynucleotide. In certain such embodiments, the label is attached to the oligonucleotide or polynucleotide via at least one linker moiety. In one aspect, the invention provides a method of preparing an oligonucleotide or polynucleotide. The method comprises (a) providing a nucleic acid synthesis reagent comprising the formula:

wherein Z is O or CH₂(ii) a B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof; BG is a blocking group; r is O, NH or S; PG is a protecting group; x is a nucleotide or nucleotide analog; n is an integer greater than 0;represents a single bond or a double bond. In addition, the method further comprises (b) removing PG from the nucleic acid synthesis reagent to produce an oligonucleotide comprising the formula:

wherein R is₁H, OH, a hydrophilic group or a hydrophobic group; thereby preparing the oligonucleotide or polynucleotide. In certain embodiments, the method comprises attaching nucleic acid synthesis reagents or components thereof (e.g., precursor reagents, such as phosphoramidites) to a solid support prior to step (b). In certain embodiments, the method comprises attaching at least one label (e.g., donor moiety, quencher moiety, acceptor moiety, etc.) to the nucleic acid synthesis reagents, components thereof, and/or the oligonucleotides or polynucleotides. In certain such embodiments, the label is attached to the nucleic acid synthesis reagent, a component thereof, and/or the oligonucleotide or polynucleotide via at least one linker moiety.

Various protecting groups are optionally used in the method of the present invention for preparing an oligonucleotide or polynucleotide. In certain embodiments, for example, the protecting group is selected from, for example, trityl, monomethoxytrityl (monomethoxytrityl), dimethoxytrityl, jerusalem artichoke glycosyl (levulinyl), fluorenylmethyloxycarbonyl (fluoromethoxycarbonyl), diphenylmethoxycarbonyl, and the like. For further example, in certain embodiments, the protecting group comprises the formula:

or

In some embodiments, the 2' -terminator nucleotide comprises the formula:

wherein R is₁H, OH, a hydrophilic group or a hydrophobic group; b is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof; BG is a blocking group; z is O or CH₂(ii) a Andrepresents a single bond or a double bond. In certain embodiments, for example, the 2 ' -terminator nucleotide comprises a 2 ' -monophosphate-3 ' -hydroxyl nucleotide. In addition, the 2' -terminator nucleotide is generally not extended by one or more nucleotide incorporating biocatalysts (nucleotidic incorporation) selected from, for example, the following: G46E E678G CS5DNA polymerase, G46E L329A E678G CS5DNA polymerase, G46E L329A D640G S671F CS5DNA polymerase, G46E L329A D640GS671 695 2E 678G CS5DNA polymerase, G46E E678G CS6DNA polymerase, Δ Z05R polymerase, E615G Taq DNA polymerase, Thermus flavus (Thermus flavus) polymerase, TMA-25 polymerase, E678G TMA-25 polymerase, TMA-30 polymerase, E678G TMA-30 polymerase, Tth DNA polymerase, Thermus SPS-17 polymerase, E615G Taq polymerase, Thermus Z05G, T7DNA polymerase, Kornberg DNA polymerase I, Klenow DNA polymerase, Kloena polymerase, Taq DNA polymerase, Taq polymerase, alpha RT polymerase, RT-DNA polymerase, RT-DNA polymerase, RT-DNA polymerase, RT-DNA polymerase, RT-, SP6 RNA polymerase, T3 RNA polymerase, T4 DNA polymerase, T7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, ribonucleotide-incorporating DNA polymerase (ribonucleotide-incorporating DNA polymerase), and the like.

In another aspect, the present invention provides a business method comprising (a) receiving an order from a customer for one or more of: (i) instructions for preparing an oligonucleotide or polynucleotide comprising a 2 '-terminator nucleotide, or (ii) an oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide. The method also includes (b) providing (i) and/or (ii) to the customer. In some embodiments, the method includes receiving the order via an electronic medium (e.g., via the Internet, etc.).

In another aspect, the invention provides a kit comprising one or more of: (a) instructions for preparing an oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide; or, (b) at least one oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide.

Brief Description of Drawings

FIGS. 1A-D schematically illustrate exemplary 2' -terminator nucleotides.

FIGS. 2A and B schematically illustrate some embodiments of 2' -terminator nucleotides.

Figures 3A-C schematically illustrate dye-labeled tetraphosphates according to various embodiments.

FIGS. 4A and B schematically illustrate certain embodiments of labeled nucleotide tetraphosphates.

FIG. 5 schematically depicts a label attached to a nucleotide tetraphosphate via a linker.

FIGS. 6A-L schematically illustrate various 2' -terminator nucleotides linked to fluorescent dyes.

Fig. 7 schematically depicts an exemplary joint.

FIG. 8 schematically illustrates a synthetic reaction to prepare a mixture of nucleotides 5 '-triphosphate-3' -monophosphate and nucleotides 5 '-triphosphate-2' -monophosphate.

FIG. 9 schematically depicts certain steps in a solid phase synthesis pathway for the preparation of uridine tetraphosphate according to one embodiment.

FIG. 10 schematically illustrates certain steps of an exemplary regiospecific (regiospecific) synthetic pathway for TAMRA-uridine tetraphosphate.

FIG. 11 schematically illustrates a polymerase bound to a template nucleic acid and to a primer nucleic acid containing an incorporated cytosine tetraphosphate nucleotide.

FIG. 12 schematically depicts the regiospecific synthetic pathway for uridine tetraphosphate.

FIG. 13 schematically illustrates a synthetic reaction to prepare a mixture of adenine nucleotides 5 '-triphosphate-3' -monophosphate and adenine nucleotides 5 '-triphosphate-2' monophosphate.

FIGS. 14A-C are HPLC profiles showing detection of adenine tetraphosphate nucleotides.

FIG. 15 schematically shows certain steps in the TAMRA-labeled uridine tetraphosphate synthesis pathway.

FIG. 16 is an HPLC chromatogram showing detection of BOC-protected uridine propargyl tetraphosphate (corresponding to structures 4 and 5 of FIG. 15).

FIG. 17 schematically illustrates certain steps in the ROX-labeled cytidine tetraphosphate synthetic pathway.

FIG. 18 schematically depicts certain steps in the R6G-labeled adenosine tetraphosphate synthesis pathway.

FIG. 19 schematically shows certain steps in the R110-labeled guanosine tetraphosphate synthesis pathway.

FIGS. 20A-D are electrophoretograms illustrating detection of multiple extended primer nucleic acids.

FIG. 21 is a map showing data of sequence analysis of an M13mp18 DNA template with 2' -terminator nucleotides.

FIGS. 22A and B are maps showing data from sequence analysis of an M13mp18 DNA template with a fluorescent dye-labeled 2' -terminator nucleotide.

FIG. 23 schematically depicts a synthetic pathway for an exemplary oligonucleotide.

FIG. 24 is a block diagram illustrating certain steps performed in a business method in accordance with one embodiment of the present invention.

FIG. 25 schematically illustrates a blocked oligonucleotide according to one embodiment of the invention.

FIG. 26 schematically illustrates a solid phase synthesis pathway for blocked oligonucleotides according to one embodiment of the invention.

FIG. 27 schematically depicts 3 '-O-TBDMS-2' -O-phosphoramidite (phosphoramidite).

FIG. 28 is a gel electrophoresis image showing detection of PCR products involved in PAP-related HIV DNA template titration analysis.

FIG. 29 is a graph depicting the threshold cycle (C) of copy number of various mutant K-Ras plasmid templates used in amplification reactions involving blocked or unblocked primers_T) The value is obtained.

FIG. 30 is a graph showing threshold cycles (C) for various enzymes and enzyme concentrations used in amplification reactions involving K-Ras plasmid templates_T) The value is obtained.

FIG. 31 is a bar graph showing the effect of enzyme concentration on threshold cycle (Ct) values in Pyrophosphorolysis Activated Polymerization (PAP) reactions using G46E L329A E678G (GLE) CS5DNA polymerase. The Y-axis represents Ct value and the X-axis represents enzyme concentration (nM). The legend indicates the number of template nucleic acids, corresponding to each column in the figure (no template nucleic acid copies (no template), 1e⁴Copied template nucleic acid (1E4/rxn), 1E⁵Copied template nucleic acids (1E5/rxn), and 1E⁶Copied template nucleic acid (1E 6/rxn)).

FIG. 32 is a bar graph showing the effect of enzyme concentration on threshold cycle (C1 value) in pyrophosphorolysis-activated polymerization (PAP) reactions using G46E L329A D640G S671F E678G (GLDSE) CS5DNA polymerase (GLDSE) the Y-axis represents Ct value, the X-axis represents enzyme concentration (nM). In the figure, the number of template nucleic acids is illustrated, corresponding to each bar in the graph (no template nucleic acid (no template), 1E⁴Copied template nucleic acids (1E4/rxn), 1E⁵Copied template nucleic acids (1E5/rxn) and 1E⁶Copied template nucleic acid (1E 6/rxn)). FIG. 33 is a bar graph showing data for PAP reverse transcription reaction of HCV RNA using quantitation specific for HCV cDNAAnd detecting the cDNA reaction product by PCR analysis. The Ct value is represented on the Y-axis and the units of enzyme used in the reaction are represented on the X-axis. As shown in the figure, the enzymes used in these reactions were a mixture of Z05 DNA polymerase (Z05) or G46E L329A Q601R D640G S671F E678G (GLQDSE) and G46E L329A Q601R D640G S671F (GLQDS) CS5DNA polymerase.

Figure 34 shows bidirectional PAP generated BRAF oncogene amplification of PCR growth curve. The X-axis shows normalized cumulative fluorescence (intensity) and the y-axis shows the cycle number of PAP PCR amplification.

Detailed Description

I. Definition of

Before the present invention is described in detail, it is to be understood that this invention is not limited to particular methods or kits, which can vary. As used in the specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a 2 '-monophosphate-3' -hydroxy nucleoside" also includes combinations of two or more 2 '-monophosphate-3' -hydroxy nucleosides. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In describing and claiming the present invention, the following terminology, and grammatical variants thereof, is used in accordance with the definitions set out below.

"2 '-terminator nucleotides" refers to nucleotide analogs that contain a Blocking Group (BG) at the 2' -position of the sugar moiety (sugar motif) of the nucleotide. "closed group" refers to usually prevent nucleic acid extension of the chemical group or part (i.e., 2' -terminator nucleotides can generally be one or more nucleotide incorporation biological catalyst extension). That is, once the 2 '-terminator nucleotide is incorporated into a nucleic acid (e.g., at the 3' -end of the nucleic acid), the blocking group prevents further extension of the nucleic acid by at least one nucleotide incorporating biocatalyst selected from the group consisting of: for example, G46E E678G CS5DNA polymerase, G46E L329AE678G CS5DNA polymerase, G46E L329A D640 2S 671F CS5DNA polymerase, G46E L329A D640G S671F E678G CS G DNA polymerase, G46G E678G CS6DNA polymerase, Δ Z05G polymerase, E615G Taq DNA polymerase, Thermus flavus polymerase, TMA-25 polymerase, E678G TMA-25 polymerase, TMA-30 polymerase, E678G TMA-30 polymerase, Tth DNA polymerase, Thermus SPS-17 polymerase, E615G Taq polymerase, Thermus Z05G polymerase, T G DNA polymerase, Kornberg DNA polymerase I, Klinenosol DNA polymerase, Taq DNA polymerase, Micrococcus DNA polymerase, alpha-DNA polymerase, reverse transcriptase, AMLV RT-RNA polymerase, MuLV RT-RNA polymerase, RNA G RNA polymerase, RNA-G RNA polymerase, RNA RT G RNA polymerase, DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, incorporation of ribonucleotide DNA polymerase. An exemplary blocking group is a phosphate group. Other representative blocking groups are also described herein. Exemplary 2 ' -terminator nucleotides include 2 ' -monophosphate-3 ' -hydroxy-5 ' -triphosphate nucleoside and 2 ' -monophosphate-3 ' -hydroxy-5 ' -diphosphate nucleoside. Other 2' -terminator nucleotides are also described herein.

An "acceptor" moiety "or" acceptor "refers to a moiety that can accept or absorb energy transferred from an energy source. In some embodiments, the acceptor moiety can also emit energy (e.g., light, heat, etc.) upon absorption of a sufficient amount of the transferred energy. In these embodiments, the receptor is also referred to as a "reporter" or "reporter". Exemplary acceptor moieties include, but are not limited to, various fluorophores, e.g.-Red 610(LC-Red 610), LC-Red 640, LC-Red 670, LC-Red705, JA-270, CY5, CY5.5, and others.

"alcohol group" means an organic group containing at least one hydroxyl group.

"aldehyde group" means an organic group comprising the formula CHO.

"alkenyl" means a straight, branched, or cyclic unsaturated hydrocarbon moiety containing one or more carbon-carbon double bonds. Exemplary alkenyl groups include ethenyl, 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1-dimethyl-2-propenyl, 1, 2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, ethyl-2-propenyl, ethyl-2-pentenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methyl-3-pentenyl, 2-methyl-3-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1-dimethyl-2-butenyl, 1-dimethyl-3-butenyl, 1-methyl-2-pentenyl, 2-methyl-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1-dimethyl-2-butenyl, 3-butenyl, 2-penten, 1, 2-dimethyl-2-butenyl, 1, 2-dimethyl-3-butenyl, 1, 3-dimethyl-2-butenyl, 1, 3-dimethyl-3-butenyl, 2-dimethyl-3-butenyl, 2, 3-dimethyl-2-butenyl, 2, 3-dimethyl-3-butenyl, 3-dimethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1, 2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl, 1, 2-dimethyl-2-butenyl, 2-ethyl-3-butenyl, 2-methyl-2-propenyl, 2-methyl-2-butenyl, 2, 1-ethyl-2-methyl-2-propenyl group and the like. Alkenyl groups typically contain about 1 to about 20 carbon atoms, and more typically contain about 2 to about 15 carbon atoms. Alkenyl groups may be substituted or unsubstituted.

"alkenylamino" refers to an amino group that contains at least one alkenyl group.

"alkoxy" refers to an alkyl group containing an oxygen atom and includes, for example, methoxy, ethoxy, propoxy, butoxy, pentoxy, heptoxy, octoxy, and the like.

"alkyl" means a straight, branched or cyclic saturated hydrocarbon moiety and includes all positional isomers, such as methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2, 2-dimethylbutyl, 2, 3-dimethylbutyl, 3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 2-trimethylpropyl, 1, 2, 2-trimethylpropyl, 1-ethyl-1-methylpropyl and 1-ethyl-2-methylpropyl, n-hexyl, cyclohexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl, n-decyl and the like. The alkyl group typically contains from about 1 to about 20 carbon atoms, and more typically contains from about 2 to about 15 carbon atoms. Alkyl groups may be substituted or unsubstituted.

"alkylamino" refers to an amino group that contains at least one alkyl group.

"alkynyl" refers to a straight, branched, or cyclic unsaturated hydrocarbon moiety containing one or more carbon-carbon triple bonds. Representative alkynyl groups include, for example, 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-2-butynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1-dimethyl-2-butynyl, 1-dimethyl-3-butynyl, 1, 2-dimethyl-3-butynyl, 2-dimethyl-3-butynyl, 3-dimethyl-l-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl, 1-ethyl-l-methyl-2-propynyl and the like. Alkynyl groups typically contain about 1-20 carbon atoms, and more typically contain about 2-15 carbon atoms. Alkynyl groups may be substituted or unsubstituted.

"alkynyl amino" refers to contain at least one alkynyl amino.

"aryl" refers to a substituent derived from an atom or moiety of an aromatic compound. Exemplary aryl groups include, for example, phenyl, benzyl, tolyl, xylyl, and the like. The aryl group optionally includes multiple aromatic rings (e.g., diphenyl, etc.). Further, the aryl group may be substituted or unsubstituted.

"aryloxy" refers to an aryl group containing an oxygen atom and includes, for example, phenoxy, chlorophenoxy, methylphenoxy, methoxyphenoxy, butylphenoxy, pentylphenoxy, benzyloxy, and the like.

The term "attaching" refers to the covalent and/or non-covalent binding of two or more substances to each other, even if only transient binding. In some embodiments, for example, the nucleic acid synthesis reagents linked to each other are part of the oligonucleotide preparation method.

"Donor moiety" refers to a moiety that is capable of transferring, emitting or providing one or more forms of excitation energy to one or more acceptor moieties.

"ester group" refers to a class of organic compounds comprising the general formula RCOOR ', wherein R and R' are independently selected from: alkyl, alkenyl, alkynyl, aryl, or combinations thereof.

"Ether group" refers to a straight chain, branched or cyclic moiety containing 2 carbon atoms attached to an oxygen atom. Exemplary ether groups include, for example, methoxymethyl, methoxyethyl, methoxypropyl, ethoxyethyl, and the like.

"halo" refers to a group containing a halogen atom, such as F, Cl, Br, or I.

"heterocycle" means a saturated, unsaturated, or aromatic monocyclic or bicyclic ring which contains one or more heteroatoms independently selected from nitrogen, oxygen, and sulfur. The heterocyclic ring may be attached to the sugar moiety of the nucleotide of the invention or an analogue thereof via any heteroatom or carbon atom. Exemplary heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactanyl, oxiranyl, oxetanyl (oxyethanyl), tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, furanyl, benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridinyl, quinolinyl, isoquinolinyl, oxazolyl, isoxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, cinnolinyl, 2, 3-diazanaphthyl, quinazolinyl, and the like.

"homocyclic" refers to a saturated or unsaturated (but non-aromatic) carbocyclic ring, such as cyclopropane, cyclobutane, cyclopentane, cyclohexane, cycloheptane, cyclohexene, and the like.

"label" refers to a moiety that is attached (covalently or non-covalently) or capable of being attached to a molecule, which moiety provides or is capable of providing information about the molecule (e.g., descriptive, identifying, etc., information about the molecule). Exemplary labels include fluorescent labels, non-fluorescent labels, colorimetric labels, chemiluminescent labels, bioluminescent labels, radioactive labels, mass modifying groups, antibodies, antigens, biotin, haptens, and enzymes (including, e.g., peroxidase, phosphatase, etc.).

"moiety" or "group" refers to one of the moieties (e.g., functional groups, substituents, etc.) into which something (e.g., a molecule) is or can be divided. For example, in some embodiments, the oligonucleotides described herein include at least one donor moiety and/or at least one acceptor moiety.

"non-extendible" nucleotide refers to a nucleotide that, upon incorporation into a nucleic acid, prevents further extension of the nucleic acid by at least one biocatalyst.

The term "nucleic acid" or "polynucleotide" refers to a nucleic acid that can be linked to ribonucleic acid (RNA) orPolymers corresponding to polymers of deoxyribonucleic acid (DNA) or analogs thereof. This includes polymers of nucleotides (e.g., RNA and DNA), as well as modified forms thereof, Peptide Nucleic Acids (PNAs), Locked Nucleic Acids (LNA)^TM) And the like. In some embodiments, the nucleic acid can be a polymer comprising multiple monomer types (e.g., RNA and DNA subunits). The nucleic acid can be or can include a chromosome or chromosome segment, a vector (e.g., an expression vector), an expression cassette, a naked DNA or RNA polymer, a product of a Polymerase Chain Reaction (PCR), an oligonucleotide, a probe, a primer, and the like. The nucleic acid can be, for example, single-stranded, double-stranded, triple-stranded, etc., and is not limited to any particular length. Unless otherwise indicated, a particular nucleic acid sequence optionally comprises or encodes a complementary sequence in addition to any sequence explicitly indicated.

Nucleic acids are not limited to molecules having naturally occurring polynucleotide sequences or structures, naturally occurring backbones, and/or naturally occurring internucleotide linkages. For example, nucleic acids containing one or more carbocyclic sugars are also included in this definition (Jenkins et al (1995)Chem.Soc.Rev.pp 169-176). To further illustrate, although nucleic acids will typically contain phosphodiester linkages, in some instances nucleic acid analogs with alternative backbones are included. These include, but are not limited to, phosphoramides (Beaucage et al (1993)Tetrahedron49(10): 1925 and references therein; letsinger (1970)J. Org.Chem.35: 3800; sprinzl et al (1977)Eur.J.Biochem.81: 579; letsinger et al (1986)Nucl.Acids Res.14: 3487; sawai et al (1984)Chem. Lett.805; letsinger et al (1988)J.Am.Chem.Soc.110: 4470; and Pauwels et al (1986)Chemica Scripta26: 1419) thiophosphate (Mag et al (1991)Nucleic Acids Res.19: 1437 and U.S. Pat. No. 5,644,048), dithiophosphates (Briu et al (1989)J.Am.Chem.Soc.111: 2321) an O-methylphosphonite (O-methylphosphonodiate) bond (Eckstein,oligo nucleotides and Analogues: a. the Practical ApproachOxford University Press (1992)), and peptide nucleic acid backbones and linkages (Egholm (1992)J.Am.Chem.Soc.114: 1895; meier et al (1992)Chem.Int. Ed.Engl.31：1008；Nielsen(1993)Nature365: 566; and Carlsson et al (1996)Nature380: 207). Other analog nucleic acids include those having a positively charged backbone (Denpcy et al (1995)Proc.Natl.Acad.Sci.USA92: 6097) non-ionic backbones (U.S. patent nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141, and 4,469,863; angew (1991)Chem. Intl.Ed.English30: 423; letsinger et al (1988)J.Am.Chem.Soc.110: 4470; letsinger et al (1994)Nucleosides& Nucleotide13: 1597; chapters 2 and 3, ASC Symposium Series580, "Carbohydrate Modifications in antisense research", ed.y.s.sanghvi and p.dan Cook; mesmaeker et al (1994)Bioorganic & Medicinal Chem.Lett.4: 395; jeffs et al (1994)J. Biomolecular NMR 34：17；Tetrahedron Lett.37: 743(1996)) and non-ribose backbones, including those described in U.S. patent nos. 5,235,033 and 5,034,506, and ASC Symposium Series580, Carbohydrate Modifications in Antisense Research, ed.y.s.sanghvi (chapters 6 and 7), and p.dan Cook. Several nucleic acid analogs are also described, for example, in Rawls,C & E Newsjun.2, 1997 page 35. Modifications of the ribose-phosphate backbone can be made to facilitate the addition of other moieties (e.g., labeling moieties), or to alter the stability and half-life of such molecules in physiological environments.

In addition to naturally occurring heterocyclic bases typically found in nucleic acids (e.g., adenine, guanine, thymine, cytosine, and uracil), nucleic acid analogs also include those having non-naturally occurring heterocyclic or other modified bases. For illustration, certain bases used in nucleotides as melting temperature (Tm) modifiers are optionally included. Some examples of these include 7-deazapurines (e.g., 7-deazaguanine, 7-deazaadenine, etc.), pyrazoles [3, 4-d]Pyrimidine, propynyl-dN (for example, propynyl-dU, propynyl-dC) and the like. See, for example, U.S. Pat. No. 5,990,303 issued to Seela on 23.11.1999, entitled "SYNTHESIS OF 7-DEAQA-2' -DEOXYGUANOSINE NUCLEOTIDE". Other representative heterocyclic bases include, for example, hypoxanthine, inosine, xanthine; 2-aminopurine, 2, 6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and 8-aza derivatives of xanthine; adenine, guanine, 2-aminopurine, 2, 6-diaminopurine, 2-amino-6-chloropurine, hypoxanthine, inosine and 7-deaza-8-aza derivatives of xanthine; 6-azacytosine; 5-fluorocytosine; 5-chlorocytosine; 5-iodocytosine; 5-bromocytosine; 5-methylcytosine; 5-propynyl cytosine; 5-bromovinyluracil; 5-fluorouracil; 5-chlorouracil; 5-iodouracil; 5, bromouracil; 5-trifluoromethyl uracil; 5-methoxymethyluracil; 5-ethynyluracil; 5 propynyluracil, and the like. Many non-naturally occurring bases are also in Seela et al (1991)Helv.Chim Acta74: 1790, Grein et al (1994)Bioorg.Med.Chem.Lett.4: 971-Helv.Chim.Acta82: 1640, and the like.

Other examples OF MODIFIED bases AND nucleotides are described, for example, IN U.S. Pat. No. 5,484,908 entitled "OLIGO nucleotide CONTAINING 5-PROPYNYL PYRIMIDES" issued to Froehler et al on 16.1.1996, U.S. Pat. No. 5,645,985 issued to Froehler et al on 8.7.1997, U.S. Pat. No. ENHANCEDTRIPLE-HESKIX AND DOUBLE-HELIX FOATION WITHALOMERS CONTAININING MODIFIED PYRIMIDES "issued to Froehler et al on 3.1998, U.S. Pat. No. 5,830,653 issued to Froehler et al on 3.11.1998, U.S. Pat. No. 2.2.1 AINIMODIFIED PYRIMIDES," U.S. Pat. No.6,639,059 issued to Kochhike et al on 28.2003, U.S. Pat. No. 2.2.1. CLOSURE. 2.1. AINIMODIFIEM MODIFIED. 2, U.16. SENTIANCE. PATENT APPLICATION, COMPLEMENTS. 15, SUMMARY FOR COMPLEPHONE MULTIPOIDESTIONATION, COMPLEON, NATION MULTIPLE.16,16, it is titled "SYNTHESIS OF [2.2.1] BICYCLO NUCLEOSIDES".

"nucleic acid synthesis reagents" refers to compounds that can be used to synthesize oligonucleotides or other nucleic acids.

"nucleoside" refers to a nucleic acid component (e.g., comprising at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, etc.) comprising a base or basic group covalently bonded to a sugar moiety (e.g., ribose, etc.), a derivative of a sugar moiety, or a functional equivalent of a sugar moiety (e.g., an analog, such as a carbocyclic ring). For example, when a nucleoside comprises a sugar moiety, the base is typically bound to the 1' -position of the sugar moiety. As noted above, the base may be naturally occurring (e.g., a purine base, such as adenine (A) or guanine (G), a pyrimidine base, such as thymine (T), cytosine (C), or uracil (U)) or non-naturally occurring (e.g., a 7-deazapurine base, a pyrazolo [3, 4-d ] pyrimidine base, a propynyl-dN base, and the like). Exemplary nucleosides include ribonucleosides, deoxyribonucleosides, dideoxyribonucleosides, carbocyclic nucleosides, and the like.

"nucleotide" refers to an ester of a nucleoside, for example, a phosphate ester of a nucleoside. For example, a nucleotide can comprise 1, 2, 3, or more phosphate groups covalently bound to the sugar moiety (5 ' position, 3 ' position, 2 ' position, etc.) of the nucleoside.

"nucleotide incorporation biocatalysts" refers to a catalyst that catalyzes the incorporation of nucleotides into nucleic acids. Nucleotide incorporation biocatalysts are typically enzymes. An "enzyme" is a protein-based and/or nucleic acid-based catalyst that acts to reduce the activation energy of a chemical reaction involving other compounds or "substrates". "nucleotide incorporating enzyme" refers to an enzyme that catalyzes the incorporation of nucleotides into nucleic acids, for example, during nucleic acid amplification processes and the like. Exemplary nucleotide incorporating enzymes include, for example, polymerases, terminal transferases, reverse transcriptases, telomere terminal transferases, polynucleotide phosphorylases, and the like. "thermostable enzyme" refers to an enzyme that is thermostable, thermotolerant, and retains sufficient catalytic activity when subjected to elevated temperatures for a selected period of time. For example, a thermostable polymerase retains sufficient activity to effect subsequent primer extension reactions when subjected to elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Heating conditions necessary FOR the denaturation of NUCLEIC ACIDs are well known to those skilled in the art and are exemplified by U.S. Pat. No. 4,683,202 entitled "PROCESS FOR AMPLIFYING NUCLEIC ACID SEQUENCES" issued to Mullis at 28.7.1987 and U.S. Pat. No. 4,683,195 entitled "PROCESS SFOR AMPLIFYING, DETECTING, AND/OR-CLONING NUCLEIC ACID SEQUENCES" issued to Mullis et al at 28.7.1987, also see U.S. Pat. No. 4,965,188. Further described, a "thermostable polymerase" refers to an enzyme suitable for use in temperature cycling reactions, such as the polymerase chain reaction ("PCR"). For a thermostable polymerase, enzymatic activity refers to catalyzing the combination of nucleotides in an appropriate manner to form a primer extension product that is complementary to a template nucleic acid.

"oligonucleotide" refers to a nucleic acid comprising at least 2 nucleic acid monomer units (e.g., nucleotides), typically more than 3 monomer units, and more typically greater than 10 monomer units. The precise size of the oligonucleotide will generally depend on a variety of factors, including the ultimate function or use of the oligonucleotide. Typically, nucleoside monomers are linked by phosphodiester linkages or analogs thereof, including phosphorothioate, phosphorodithioate, phosphoroselenoate (phosphoroselenoate), phosphorodiselenoate (phosphorodiselenoate), phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like, including bound counterions, e.g., H⁺、NH₄ ⁺、Na⁺Etc., if such counterions are present. Optionally, oligonucleotides are prepared by any suitable method, including, but not limited to, isolation of existing or native sequences, DNA replication or amplification, reverse transcription, cloning and restriction digest of appropriate sequences, or by direct chemical synthesis, e.g., Narang et al (1979)Meth.Enzymol.68: 90-99 phosphotriester process; brown et al (1979)Meth.Enzymol.68: 109-151 phosphodiester method; beaucage et al (1981)Tetrahedron Lett.22: 1859 the diethylphosphoramidite method of 1862; matteucci et al (1981)J.Am.Chem.Soc.103: 3185 and 3191; an automated synthesis method; or in 1984U.S. Pat. No. 4,458,066 entitled "PROCESS FOR PREPARING POLYNUCLEOTIEDES" issued on 3/7.M. to Caruthers et al, or other methods known to those skilled in the art.

"phosphoramidite" refers to a compound containing a group comprising the formula:

wherein R is₁And R₂Is an alkyl group independently selected from the group consisting of: methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and heptyl; r₃Is (CH)₂)₂CN or CH₃. In certain embodiments, for example, the phosphoramidite is a nucleoside-3 '-phosphoramidite monomer or a nucleoside-2' -phosphoramidite monomer commonly used in oligonucleotide synthesis methods. In certain embodiments, the 5' hydroxyl position of these phosphoramidite monomers is protected with a protecting group. Different protecting groups are also typically attached to the exocyclic amine of the base. In addition, optionally with beta-cyanoethyl (R)₃) And diisopropylamine (NR)₁R₂) The radicals or other radicals corresponding to the above formulae protect the phosphorus atom of the monomers. The synthesis of phosphoramidites and oligonucleotides is also described, for example, in Beaucage et al (1992) "Advances in the synthesis of oligonucleotides by the phosphoramidite apuach"Tetrahedron48: 2223-2311.

"protecting group" refers to a chemical group that is covalently or non-covalently bound (e.g., removably bound) to a given compound, preventing an adverse chemical reaction at one or more sites of the compound. Exemplary protecting groups include trityl, monomethoxytrityl, dimethoxytrityl, Jerusalem artichoke glycosyl, fluorenylmethoxycarbonyl, benzhydryloxycarbonyl, and the like.

The term "pyrophosphorolysis" refers to the Presence of Pyrophosphate (PP)₁) Removing one from the nucleic acid in the presence ofOr multiple nucleotides to produce one or more nucleoside triphosphates.

"quencher moiety" or "quencher" refers to a moiety that reduces the emission of detectable radiation (e.g., fluorescent or luminescent radiation) from a source that would otherwise have emitted such radiation. The quencher typically reduces the detectable radiation emitted from the source by at least 50%, typically at least 80%, more typically at least 90%. Certain quenchers may re-emit energy absorbed from, for example, a fluorescent dye with a signal that is unique to the quencher, and thus the quencher may also be an acceptor moiety. This phenomenon is commonly referred to as fluorescence resonance energy transfer or FRET. Alternatively, the quencher may dissipate the energy absorbed from the fluorescent dye in a form other than light (e.g., heat). Molecules commonly used in FRET applications include, for example, fluorescein, FAM, JOE, rhodamine, R6G, TAMRA, ROX, DABCYL, and EDANS. Whether a fluorescent dye is an acceptor or a quencher is defined by its excitation and emission spectra, and the fluorescent dye with which it is paired. For example, FAM is most efficiently excited by light of 488nm wavelength and emits light in the spectrum of 500 to 650nm with a maximum emission of 525 nm. FAM is a suitable donor moiety for use with, for example, TAMRA quenchers with a maximum excitation of 514 nm. Exemplary non-fluorescent or dark Quenchers that dissipate the energy absorbed from fluorescent dyes include Black Hole Quenchers sold by Biosearch Technologies, Inc. (Novato, Calif., USA)^TM。Black Hole Quenchers^TMIs a structure comprising at least 3 free radicals selected from substituted or unsubstituted aryl or heteroaryl compounds or combinations thereof, wherein at least 2 residues are bound by an exocyclic diazo bond (see, e.g., Cook et al, international publication No. WO 01/86001, entitled "darkquerches FOR dornor-accepttor ENERGY TRANSFER", published 11/15 2001). Exemplary quenchers are also provided in U.S. patent No.6,465,175 entitled "quenching quenchers catalysts quenching catalysts belts, andethods OF USE theroflef," issued to Horn et al at 10, 15, 2002.

"silyl" is meant to include the formula SiRR₁R₂A compound of the class wherein R, R₁And R₂Independently H, alkyl, alkenyl, alkynyl, aryl, or combinations of these groups.

"solid support" refers to a solid material that can be derivatized with, or bound to, a chemical moiety, such as a probe or the like. Exemplary solid supports include plates, beads, microbeads, tubes, fibers, whiskers (whisker), combs, hybridization chips (including microarray plates, e.g.Those used in probe detection (Affymetrix, inc., Santa Clara, CA, usa), etc.), films, single crystals, ceramic layers, self-assembled monolayers, etc.

"terminator nucleotides" refers to incorporation of nucleic acid after the incorporation of nucleic acid by at least one nucleotide incorporation biological catalyst to prevent further extension of nucleic acid nucleotide.

"thioether group" means a straight, branched, or cyclic moiety comprising 2 carbon atoms bonded to a single sulfur atom, including, for example, methylthiomethyl, methylthioethyl, methylthiopropyl, and the like.

Review of

The present invention relates to blocked oligonucleotides that can be used as probe and/or primer nucleic acids in a variety of nucleic acid technologies. For example, the blocked oligonucleotides described herein can be readily substituted in a variety of PCR-related methods without sacrificing their ease of use, and from an economic standpoint, they can replace previously existing blocked oligonucleotides. Some applications of these blocked oligonucleotides are further illustrated in the examples provided below and are described, for example, in U.S. patent application No.10/879493 entitled "2' -TERMINATOR RELATED PYPHOSPHOROSIS ACTIVATED POLYMERIZATION" filed on 6.2004 by Gelfand et al and U.S. patent application No.60/852882 entitled "MUTANT DNAPOLYMERASES AND RELATED METHODS" filed on 18.2006 by Bauer et al.

The blocked oligonucleotides and polynucleotides of the invention contain a 2 '-terminator nucleotide, which 2' -terminator nucleotide renders the oligonucleotides and polynucleotides unextended by various nucleotide incorporation biocatalysts. The 2 '-terminator nucleotides of these oligonucleotides and polynucleotides typically have an intact sugar ring or sugar-like ring (e.g., carbocyclic ring, etc.), and contain a blocking group (e.g., negatively charged blocking group, bulky blocking group, etc.) at the 2' -position of these sugar moieties. In addition to methods of making these oligonucleotides and polynucleotides, the invention also provides related commercial methods and kits. These and many other features of the present invention are described further below.

III.2' -terminator nucleotides

The present invention relates to methods for preparing oligonucleotides and polynucleotides comprising 2' -terminator nucleotides. Oligonucleotide synthesis and related nucleic acid synthesis reagents are further described below. Nucleotides used in various embodiments of the invention generally include a hydroxyl group at the 3 '-position of the intact sugar ring and a blocking group (e.g., negatively charged blocking groups, bulky blocking groups, etc.) at the 2' -position of the sugar moiety. Certain biocatalysts described herein comprise the ability to extend primer nucleic acids with these 2' -terminator nucleotides in a template-directed manner. After the 2 '-terminator nucleotide is incorporated into the primer nucleic acid 3' -terminal, generally rendering the nucleic acid can not be extended by a biocatalyst. In addition, some biocatalysts include the ability to remove 2' -terminator nucleotides from oligonucleotides by, for example, pyrophosphorolysis. Thus, the oligonucleotides of the invention are also optionally used as primer nucleic acids in various PAP applications. Certain applications of the blocked oligonucleotides of the invention and nucleotide incorporation biocatalysts for use in these applications are also described, for example, in U.S. Pat. No.10/879,493 to Gelfand et al, 6.2004, entitled "2' -TERMINATERERATED PYROPHOSPHOROLYSIS ACTIVATED POLYMERIZATION", and U.S. patent application No.60/852882 to Bauer et al, 2006, 10.18.10.2004, entitled "MUTANT DNA POLYMERASES AND RELATED METHODS". Additional details regarding 2 '-TERMINATOR nucleotides and nucleosides can be found, for example, in U.S. patent application No.10/879,493, entitled "2' -TERMINATOR RELATED PYROPHOSPHOROLYSIS ACTIVATED POLYMERIZATION", filed 6/28/2004 by Gelfand et al.

2' -terminator nucleotides used in the methods and other aspects of the invention generally include the formula:

wherein R is₁H, OH, a hydrophilic group or a hydrophobic group; b is at least one homocyclic ring, at least one heterocyclic ring (with or without an exocyclic heteroatom), at least one aryl group, or a combination thereof; BG is a blocking group; z is O or CH₂(ii) a And isRepresents a single bond or a double bond. In some embodiments, these nucleosides and nucleotides are labeled. In addition, these 2 '-terminator nucleotides generally contains 1, 2, 3 or more connected to the 5' position of the phosphate group. In one embodiment, for example, the 2 '-terminator nucleotide comprises 2' -monophosphate-3 '-hydroxy-5' -triphosphate nucleoside.

FIGS. 1A-D schematically illustrate some embodiments of 2' -terminator nucleotides. Specifically, FIG. 1A schematically shows an adenosine tetraphosphate terminator nucleotide, FIG. 1B schematically depicts a guanosine tetraphosphate terminator nucleotide, FIG. 1C schematically illustrates an uridine tetraphosphate terminator nucleotide, and FIG. 1D schematically shows a cytidine tetraphosphate terminator nucleotide.

A. Base

At the 1 'position of the sugar moiety of a 2' -terminator nucleoside or nucleotide, it optionally contains substantially any heterocyclic or aryl group (i.e., as a base or B group) that can base pair with another nucleic acid, e.g., by hydrogen bonding or base stacking mechanisms. Thus, no attempt is made herein to describe all possible groups that may be used. However, for illustrative purposes, certain representative B groups are provided below. In some embodiments, for example, B comprises the formula:

wherein X₁And X₂Independently selected from CR₈Or N; r₂Is H, OH or NR₄R₅；R₃Is H, OH or NR₆R₇；R₄、R₅、R₆And R₇Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, aryloxy, and combinations thereof; r₈Is H, halo, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, alkylol, alkenylalcohol, alkynylalcohol, unsubstituted polyethylene glycol or substituted polyethylene glycol.

In other embodiments, B comprises the formula:

wherein X₁And X₂Independently selected from CH and N; r₂Is O or S; r₃Is H, OH or NR₄R₅；R₄And R₅Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, and combinations thereof.

In some embodiments, B comprises the formula:

wherein R is₂Is H, OH or NR₄R₅；R₃Is H, OH or NR₆R₇；R₄、R₅、R₆And R₇Independently of each otherSelected from the group consisting of H, alkyl, alkenyl, alkynyl, benzyl, aryl, and combinations thereof.

In some embodiments, B comprises the formula:

wherein X is CH or N; r₂And R₃Independently selected from H, OH and NHR₄；R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; r₅Is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino or combinations thereof.

In other embodiments, B comprises the formula:

wherein X is CH or N; r₂Is O or S; r₃Is H, OH or NHR₄；R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; and R is₅Is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino or combinations thereof.

In certain embodiments, B comprises the formula:

wherein X₁And X₂Independently selected from CH and N; r₂Selected from H, alkyl, alkenyl, benzyl, aryl, aryloxy, or combinations thereof; and R is₃Is O or S.

In other embodiments, B comprises the formula:

wherein R is₂And R₃Independently selected from O and S; and R is₄And R₅Independently selected from H, NH₂SH, OH, alkyl, alkenyl, alkynyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof.

In some embodiments, B comprises the formula:

wherein R is₂And R₃Independently selected from O and S; and R is₄Is H, NH₂SH, OH, alkyl, alkenyl, benzyl, aryl, aryloxy, alkoxy, halo, or combinations thereof.

In other embodiments, B comprises the formula:

wherein R is₂And R₃Independently selected from O and S.

In some embodiments, B comprises the formula:

wherein R is₂And R₃Independently selected from O and S, R₄Is H, alkyl, alkenyl or alkynyl.

In other embodiments, B comprises the formula:

wherein R is₂Is O or S; r₃And R₄Independently selected from H, NH₂、SH、OH、COOH、COOCH₃、COOCH₂CH₃、CHO、NO₂CN, alkyl, alkenyl, alkynyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof; r₅Is alkyl, alkenyl, aryl, benzyl, or combinations thereof.

B. Blocking group

The Blocking Group (BG) used at the 2' position of the sugar moiety also includes various embodiments. In some embodiments, for example, BG is a negatively charged group and/or a bulky group. Further illustratively, BG is optionally selected from, e.g., CN, NO₂、N₃Halogen group, ether group, alkyl ether group, aryl ether group, aldehyde group, carboxylic acid group, ester group, amino group, OCH₃、OCH₂COOH, O-silyl ether groups, ketone groups, O-lactone groups, O-alkyl groups, O-cycloalkyl groups, O-alkenyl groups, O-alkynyl groups, carbamate groups, imide groups, amide groups, and combinations thereof. More specifically, BG optionally comprises the formula:

in other embodiments, BG comprises the formula:

wherein Q is O, S or NH; x is H, OH, CH₃、BH₃F or SeH; and Z is O, S or Se. FIG. 2B schematically depicts a nucleotide comprising a blocking group having this formula. Further illustratively, BG optionally comprises the formula:

wherein Q is O, S or NH; x is O, S or NH; z is O, S or Se; r is alkyl, alkenyl or alkynyl. FIG. 2A schematically depicts a 2' -terminator nucleotide comprising a blocking group having this formula. In another exemplary embodiment, BG comprises the formula:

wherein Q is O, S or NH; x is O, S or NH; z is O, S or Se; l is-CONH (CH)₂)_nNH-、-CO(CH₂)_nNH-or-CONH (CH)₂CH₂O)_nCH₂CH₂NH-; n is an integer greater than 0; and R is NH₂SH, COOH, quencher moieties, reporter moieties, biotin or affinity moieties.

C. Marking

The oligonucleotides and polynucleotides described herein are optionally labeled after synthesis. In some embodiments, nucleic acid synthesis reagents (e.g., phosphoramidite precursors of 2' -terminator nucleotides, phosphoramidite precursors of other nucleotides, oligonucleotides and polynucleotides comprising protecting groups, etc.) are labeled prior to synthesis of oligonucleotides and polynucleotides. For example, the label is optionally attached to such as 2' -terminator nucleotides or other nucleotides homologous ring, heterocyclic or aryl (for example, through pyrimidine C⁵Cytidine N⁴N of purine⁷N of adenosine⁶C of purine⁸Or another binding site known in the art), e.g.,by amide, ester, thioester, ether, thioether, carbon-carbon, or other type of covalent bond. In addition, or alternatively, the label is bound to the sugar moiety (e.g., ribose, etc.) of the 2 '-terminator nucleotide or other nucleotide (e.g., dNTP, etc.) or an analog thereof (e.g., carbocyclic ring, etc.), and/or the phosphate group of the 2' -terminator nucleotide or other nucleotide, which can be bound, for example, by a covalent bond that is an amide, ester, thioester, ether, thioether, carbon-carbon, or other bond. Covalent bonds are typically formed in the reaction between the label and the electrophilic and nucleophilic groups of the nucleotide. In certain embodiments, the label and the nucleotide are conjugated directly to each other (e.g., via a carbon-carbon single bond, a carbon-carbon double bond, a carbon-carbon triple bond, or an aromatic carbon-carbon bond, or via a carbon-nitrogen bond, a nitrogen-nitrogen bond, a carbon-oxygen bond, a carbon-sulfur bond, a phosphorous-oxygen bond, a phosphorous-nitrogen bond, etc.). Optionally, the linker binds the label to the 2' -terminator nucleotide or other nucleotides. A wide variety of linkers can be used, or can be adapted for use in conjugating labels and nucleotides. Certain non-limiting explanations of such linkers are mentioned herein.

For further illustration, FIGS. 3A-C schematically illustrate dye-labeled tetraphosphates according to certain embodiments of the invention. Specifically, figure 3A schematically illustrates a linker group to 2 ' -terminator nucleotides in the base of the reporter dye, figure 3B schematically described through the linker group to 2 ' -terminator nucleotides in the closed group of the reporter dye, figure 3C schematically illustrated through the linker group to 2 ' -terminator nucleotides in the sugar moiety of the reporter dye, wherein X is H, OH, NHR₁、SR₁Alkyl, benzyl, aryl, alkyl-aryl, alkenyl, alkynyl, alkoxy, and the like (wherein R is₁Is H, alkyl, benzyl, aryl, alk-aryl, alkenyl, alkynyl, etc.), OR contains O, S, N, C, etc., Y is OR₂、SR₂、NHR₂Etc. (wherein R is₂Is H, alkyl, alkenyl, alkynyl, aryl, alk-aryl, etc.), Z contains O, S, N, C, Si, etc. FIGS. 4A and B also schematically illustrate some embodiments of labeled nucleoside tetraphosphates. Specifically, FIGS. 4A and B are schematic representations ofLabels bound to the base of nucleoside tetraphosphates via a linker are known, where R is selected from, for example, H, OH, alkyl, aryl, alk-aryl, alkenyl, alkynyl, and the like. Furthermore, fig. 5 schematically depicts a label bound to the phosphate group of a nucleoside tetraphosphate via a linker. FIGS. 6A-L also schematically illustrate various 2' -terminator nucleotides with fluorescent dyes that bind to the base of the nucleotide, according to some embodiments. Specifically, FIGS. 6A-C schematically illustrate R6G-labeled adenosine tetraphosphate, FIGS. 6D-F schematically illustrate R110-labeled guanosine tetraphosphate, FIGS. 6G-I schematically illustrate TAMRA-labeled uridine tetraphosphate, and FIGS. 6J-L schematically illustrate ROX-labeled cytidine tetraphosphate. Of course, as described herein, labels can be attached to 2' -terminator nucleotides or other nucleotides at other positions, including through linker binding. For purposes of illustration, FIG. 7 schematically depicts one embodiment of a joint. In some embodiments, for example, the 2' -terminator nucleotides of figures 6C, 6F, 6I and 6L contain the linker of figure 7.

Essentially any label is optionally used to label the nucleotides and nucleosides used in the oligonucleotides described herein. In some embodiments, for example, the label comprises a fluorescent dye (e.g., a rhodamine dye (e.g., R6G, R110, TAMRA, ROX, etc.), a fluorescein dye (e.g., JOE, VIC, TET, HEX, FAM, etc.), a halofluorescein dye, a cyanine dye (e.g., CY3, CY3.5, CY5, CY5.5, etc.),dyes (e.g., FL, 530/550, TR, TMR, etc.), ALEXADyes (e.g., 488, 532, 546, 568, 594, 555, 653, 647, 660, 680, etc.), dichlororhodamine dyes, energy transfer dyes (e.g., BIGDYE)^TMv 1 dye, BIGDYE^TMv 2 dye, BIGDYE^TMv 3 dyes, etc.), fluorescent (Lucifer) dyes (e.g., Lucifer yellow, etc.), CASCADEOregon Green, and the like. Additional details regarding fluorescent dyes are provided in, for example, Haugland,Molecular Probes Handbook of Fluorescent Probes and Research Products9 th edition (2003) and updates thereof. Fluorescent dyes are generally readily available from various commercial suppliers, including, for example, Molecular Probes, Inc. (Eugene, OR, USA), Amersham Biosciences Corp. (Piscataway, NJ, USA), Applied Biosystems (Foster City, Calif., USA), and the like. Other labels include, for example, biotin, a weak fluorescent label (Yin et al (2003)Appl Environ Microbiol.69(7): 3938, Babendare et al (2003)Ahal.Biochem.317(1): 1, and Jankowiak et al (2003)Chem Res Toxicol.16(3): 304) non-fluorescent markers, colorimetric markers, chemiluminescent markers (Wilson et al (2003)Analyst.128(5): 480 and Roda et al (2003)Luminescence18(2): 72) raman labeling, electrochemical labeling, bioluminescent labeling (Kitayama et al (2003)Photochem Photobiol.77(3): 333, Arakawa et al (2003)Anal.Biochem.314(2): 206, and Maeda (2003)J.Pharm.Biomed.Anal.30(6): 1725) alpha-methyl-PEG labeling reagents, for example, are described in U.S. provisional patent application No.60/428,484, filed 11/22/2002.

In certain embodiments, the label comprises a radioisotope, e.g.³H、¹⁴C、²²Na、³²P、³³P、³⁵S、⁴²K、⁴⁵Ca、⁵⁹Fe、¹²⁵I、²⁰³Hg, and the like. For further illustration, the label also optionally includes at least one mass-modifying group. For example, the mass modifying group is optionally selected from, for example, deuterium, F, Cl, Br, I, S, N₃、XY、CH₃、SPO₄、BH₃、SiY₃、Si(CH₃)₃、Si(CH₃)₂(C₂H₅)、Si(CH₃)(C₂H₅)₂、Si(C₂H₅)₃、(CH₂)_nCH₃、(CH₂)_nNY₂、CH₂CONY₂、(CH₂)_nOH、CH₂F、CHF₂、CF₃And a phosphorothioate group, wherein X is O, NH, NY, S, NHC (S), OCO (CH)_nCOO、NHCO(CH₂)_nCOO、OSO₂O、OCO(CH₂)_n、NHC(S)NH、OCO(CH₂)_nS、OCO(CH₂)S、NC₄O₂H₂S, OPO (O-alkyl) or OP (O-alkyl); n is an integer from 1 to 20, including 1 and 20; y is H, deuterium, alkyl, alkoxy, aryl, paraformaldehyde, monoalkylated paraformaldehyde, polyethyleneimine, polyamide, polyester, alkylated silyl, heterooligo (heterooligo), polyamino acid, heterooligo/polyamino acid group or polyethylene glycol group. Additional details regarding nucleic acid labeling and Sequence analysis are provided, for example, in Sterky et al (2000) "Sequence analysis of genes and genes"J.Biotech.76(2000)：1，Sensen(Ed.)Biotechnology，Volume 5B，Genomics and Bioinformatics，John Wiley &Sons, Inc. (2001), and Sensen (Ed.)Essentials of Genomics and Bioinformatics，John Wiley & Sons，Inc.(2002)。

A variety of linkers can be used to bind the label to the nucleic acid, and will be well known to those skilled in the art. Linkers generally have a structure that is sterically and electronically suitable for incorporation into nucleic acids. Linkers optionally include, for example, ether, thioether, carboxamide, sulfonamide, urea, urethane, hydrazine, or other moieties. To further illustrate, the linker typically comprises from about 1 to about 25 non-hydrogen atoms selected from, for example, C, N, O, P, Si, S, and the like, and comprises substantially any combination of the following bonds: for example, ether, thioether, amine, ester, carboxamide, sulfonamide, hydrazide linkages, and aromatic or heteroaromatic linkages. In some embodiments, the linker comprises a combination of a single carbon-carbon bond and a carboxamide or thioether bond. Although longer linear segments of linkers are optionally used, the longest linear segment generally contains from about 3 to about 15 non-hydrogen atoms, including 1 or more heteroatoms.

Non-limiting examples of linker moieties include substituted (e.g., functionalized) or unsubstituted groups such as polyethylene glycol (PEG), polymethylene, arylene, alkylarylene groups, arylenealkyl, arylthio, amidoalkyl, alkynylalkyl, alkenylalkyl, alkyl, alkoxy, sulfur groups, aminoalkyl, morpholine-derived phosphate esters, peptide nucleic acids (e.g., N- (2-aminoethyl) glycine, and the like), and the like. Some further descriptions of these and other linkers are found, for example, in U.S. Pat. No.6,339,392 to Haughland et al, Hobbs, Jr. et al, U.S. Pat. No. 5,047,519 to Iizuka et al, U.S. Pat. No. 4,711,958 to Stavrianopoulos, U.S. Pat. No. 5,175,269 to Stavrianopoulos, U.S. Pat. No. 4,711,955 to Ward et al, U.S. Pat. No. 5,241,060 to Engelhardt et al, U.S. Pat. No. 5,328,824 to Ward et al, and U.S. Pat. Pub. No. 2002/0151711 to Khan et al. Additional details regarding nucleic acid labels and linkers are provided in, for example, Hermanson,Bioconiugate Techniqueselsevier Science (1996). In certain embodiments, suitable linkers comprise photocleavable moieties, such as 2-nitrobenzyl moieties, α -substituted 2-nitrobenzyl moieties (e.g., 1- (2-nitrophenyl) ethyl moieties), 3, 5-dimethoxybenzyl moieties, thiohydroxamic acids, 7-nitroindoline moieties, 9-phenylxanthyl moieties, benzoin moieties, hydroxybenzoylmethyl moieties, NHS-ASA moieties, and the like. Photocleavable linkers are further described, for example, in U.S. patent publication No. 2003/0099972 to Olejnik et al. In some embodiments, the linker comprises a metal, such as a platinum atom. These are further described, for example, in U.S. Pat. No. 5,714,327 to Houthoff et al. Many different lengths of linker are commercially available from various suppliers, including, for example, Qiagen-Operon Technologies, Inc. (Alameda, CA, USA), BD Biosciences Clontech (Palo Alto, CA, USA), and Molecular Biosciences (Boulder, CO, USA).

IV.2' -terminator nucleoside and nucleotide Synthesis

The 2' -terminator nucleosides and nucleotides contained in the oligonucleotides of the present invention can be synthesized using various methods. For example, a method of preparing a labeled, inextensible nucleotide comprises linking at least one phosphate group to the 5 '-position of the sugar moiety of a nucleoside (e.g., a ribonucleoside, a carbocyclic nucleoside, etc.), and linking at least one blocking group to the 2' -position of the sugar moiety of the nucleoside. Exemplary blocking groups and bases optionally included in the nucleosides used in the methods are described herein. The method also includes attaching at least one label to the sugar moiety, blocking group, and/or base of the nucleoside. Suitable labels are described further below.

To further illustrate, an optionally used method of preparing a nucleoside 2 '-monophosphate comprises reacting a nucleotide comprising the formula with trisodium trimetaphosphate (NaPO) under conditions effective to produce the nucleoside 2' -monophosphate₃)₃Reaction:

wherein P is at least one phosphate group; n is an integer greater than 0; r₁H, OH, a hydrophilic group or a hydrophobic group; b is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof; z is O or CH₂(ii) a And isRepresents a single bond or a double bond. In certain embodiments, for example, the nucleotide comprises 2 phosphate groups, while in other embodiments, the nucleotide comprises 3 phosphate groups or more. Effective conditions for preparing nucleotides generally include conducting the reaction in a solution at basic pH. For example, the synthesis is typically performed at a pH greater than about 8.0, more typically greater than about 10.0, and more typically greater than about 12.0 (e.g., about 12.5, 13.0, 13.5, or 14.0). Various basic compounds can be used to adjust the pH of the reaction mixture, including, for example, KOH and NaOH, as well as many other bases widely known in the art. Nucleotides are generally limiting reagents. Although other temperature conditions are optionally used, these combinationsThe reaction is typically carried out at or near room temperature (i.e., about 20 ℃ to about 30 ℃, e.g., at about 23 ℃,24 ℃, 25 ℃,26 ℃, etc.). In addition, these reactions are typically allowed to proceed for at least about 4 hours, typically at least about 6 hours, and even more typically at least about 16 hours.

In addition, FIG. 8 schematically shows a synthetic reaction that produces a mixture of nucleotides 5 '-triphosphate-3' -monophosphate and nucleotides 5 '-triphosphate-2' -monophosphate (e.g., in a molar ratio of about 50: 50). Synthesis of a mixture of purine nucleotides is provided in one example below. Specific or at least selective synthetic pathways are also described herein. In embodiments where a mixture of nucleotides is prepared, the methods generally further comprise separating the nucleotide 5 '-triphosphate-2' -monophosphate from the nucleotide 5 '-triphosphate-3' -monophosphate. A variety of separation methods can be used to separate the nucleotide 5 '-triphosphate-2' -monophosphate from other compounds or impurities, including liquid chromatography. Various isolation techniques for purifying nucleotide synthesis products are further described, for example, in Skoog et al,Principles of Instrumental Analysis5 th edition, Harcourt Brane College Publishers (1998) and Currell,Analytical Instrumentation：Performance Characteristics and Quality，John Wiley & Sons，Inc.(2000)。

as noted above, various regiospecific or at least regioselective synthetic routes can also be used, if not completely eliminated, such that product purification is generally minimized. These synthetic routes typically include various protecting groups for the 3' -position of the sugar moiety (e.g., tert-butyldimethylsiloxane (TBDMS), SiR₁R₂R₃(wherein R is₁、R₂And R₃Independently selected from alkyl), triisopropylsilyl-oxymethyl (TOM), and the like).

The synthetic pathway of the present invention is further illustrated, for example, in FIG. 9, which schematically depicts certain steps in a solid phase synthetic pathway for uridine tetraphosphate. Furthermore, FIG. 10 schematically illustrates certain steps in the regiospecific synthesis pathway of TAMRA-uridine tetraphosphate according to an embodiment of the present invention. Other synthetic pathways and other aspects associated with the preparation of 2' -terminator nucleotides are provided in the examples below.

Various synthesis techniques are generally known, which can be applied to the synthesis method of the present invention, described in, for example, March,Advanced Organic Chemistry：Reactions，Mechanisms，and Structure，4^th Ed.，John Wiley &sons, Inc. (1992), and Carey and Sundberg,Advanced Organic Chemistry Part A：Structure and Mechanism4th Ed., Plenum Press (2000). Chemical starting materials and other reaction components useful in the synthesis of nucleotides of the invention are readily available from various commercial suppliers, including, for example, Sigma-Aldrich, Inc.

Synthesis of blocked oligonucleotides and polynucleotides comprising 2' -terminator nucleosides

The synthesis of blocked oligonucleotides and polynucleotides comprising 2' -terminator nucleosides can be achieved using various types of nucleic acid synthesis reagents. To illustrate, the nucleic acid can be synthesized enzymatically (e.g., using nucleotide incorporation biocatalysts (e.g., DNA polymerases, ligases, etc.)) or by chemical synthesis (e.g., using the phosphoramidite method or the phosphite triester method) (Herdewijn,Oligonucleotide Synthesis： Methods and Appllications，Humana Press(2005)，Gait(Ed.)，Oligonucleotide Synthesisoxford University Press (1984), Vorbruggen et al,handbook of nucleoside Synthesis，John Wiley &Sons, inc. (2001), and Hermanson,Bioconiugate Techniqueselsevier Science (1996)) to synthesize oligonucleotides. Labels can be introduced during enzymatic synthesis using, for example, labeled nucleoside triphosphate monomers (e.g., labeled extendible nucleotides, labeled 2' -terminator nucleotides, etc.), or labeled non-nucleotides or nucleotide phosphoramidites, or labels can be introduced during chemical synthesis, or labels can be introduced post-synthetically.

Enzymatic synthesis of labeled oligonucleosidesExemplary methods of acid include denaturing the template or target nucleic acid, and annealing a pair of primers to the template. In some embodiments, a mixture of deoxynucleoside triphosphates (e.g., dGTP, dATP, dCTP, and dTTP) is added to the reaction mixture, wherein at least a portion of one of the deoxynucleotides is labeled as described herein. Next, a nucleotide incorporating catalyst, such as a DNA polymerase, is added to the reaction mixture, typically under conditions that render the enzyme active. The labeled oligonucleotide is formed by incorporating labeled deoxynucleotides during polymerase chain synthesis. The DNA polymerase used in this method is generally thermostable, and the reaction temperature is typically cycled between denaturation and extension temperatures, to synthesize labeled complementary strands of the target nucleic acid by PCR (Edwards et al (Eds.),Real-Time PCR：AnEssential Guidehorizon Scientific Press (2004), Innis et al (Eds.),PCR Strategies，Elsevier Science &technology Books (1995), and Innis et al (Eds.),PCR Protocolsacademic Press (1990). Thereafter, the desired amplification product (amplicon) is separated from the other components of the reaction mixture using various purification techniques known to those skilled in the art. The amplification product can then be denatured and annealed to the template nucleic acid under conditions in which 2 '-terminator nucleotides are incorporated at the 3' ends of the individual amplification product strands to produce the desired blocked oligonucleotides. Alternatively, oligonucleotides comprising 2' -terminator nucleosides (synthesized enzymatically or chemically) can be ligated to the amplification product strands to generate the desired blocked oligonucleotides. Those skilled in the art will be aware of other variants of these enzymatic methods of synthesizing blocked oligonucleotides.

Blocked oligonucleotides prepared using chemical synthesis are typically prepared using the phosphoramidite method, although other methods are also optionally used. Phosphoramidite-based synthesis is typically performed with growing oligonucleotide chains attached to a solid support so that excess reagents in the liquid phase can be easily removed by filtration. This eliminates the need for additional purification steps between cycles.

For a brief description of an exemplary solid phase oligonucleotide synthesis cycle using the phosphoramidite method, a solid support comprising protected nucleotide monomers is typically initially treated with an acid (e.g., trichloroacetic acid) to remove the 5' -hydroxy protecting group, releasing the hydroxy group for subsequent coupling reactions. The activated intermediate is then formed, typically by the simultaneous addition of the protected phosphoramidite nucleoside monomer and a weak acid (e.g., tetrazole) to the reactants. The weak acid protonates the nitrogen of the phosphoramidite, thereby forming a reactive intermediate. The addition of nucleosides to a growing nucleic acid strand is typically completed within 30 seconds. Thereafter, a capping step is typically performed to terminate any oligonucleotide strand to which no nucleoside has been added. Capping may be performed with, for example, acetic anhydride, 1-methylimidazole, or the like. The internucleotide linkage is then converted from a phosphite to a more stable phosphotriester by oxidation using, for example, iodine as the oxidant and water as the oxygen donor. After oxidation, the hydroxyl protecting group is typically removed with a protic acid (e.g., trichloroacetic acid or dichloroacetic acid), and the cycle is repeated until chain extension is complete. After synthesis, the synthesized oligonucleotides are typically cleaved from the solid support using a base such as ammonium hydroxide or tert-butylamine. The cleavage reaction also removes any phosphate protecting groups (e.g., cyanoethyl). Finally, the protecting groups on the exocyclic amine of the base and the hydroxyl protecting groups on the one or more labeled moieties are removed by treating the oligonucleotide solution at elevated temperature (e.g., up to about 55 ℃) under basic conditions.

A chemical description FOR forming oligonucleotides by the PHOSPHORAMIDITE method is provided, FOR example, in U.S. Pat. No. 4,458,066 entitled "PROCESS FOR PREPARING POLYNUCLEOTIEDES" issued to Caruthers et al at 7/3 1984 AND in U.S. Pat. No. 4,415,732 entitled "PHOSPHORAMIDITE COMPOSITIONS AND PROCESSES" issued to Caruthers et al at 11/15 1983.

Any phosphoramidite nucleoside monomer can be labeled as desired. In certain embodiments, if the 5' -end of the oligonucleotide is to be labeled, a labeled nucleotide-free phosphoramidite can be used in the final condensation step. If the internal position of the oligonucleotide is to be labeledAlternatively, a labeled nucleotide phosphoramidite may be used during any of the condensation steps. Alternatively, oligonucleotides can be labeled at a significant number of positions after synthesis (Eckstein et al (Eds.),Oligonucleotides and Analogues：A Practical Approachoxford University Press (1992), Chu et al (1983) "Derivatification of unprotected polynuclotides"Nucleic Acids Res.11(18): 6513-6529 and U.S. patent No. 5,118,800 entitled "Oligonucleotides side passage a primary amino group in the tertiary nucleotide", issued to Smith et al on 2.6.1992. To further illustrate, it is also possible to use the phosphorus diesters on their backbone (Eckstein et al (1992), supra) or at the 3' -end (Nelson et al (1992) "oligonucleotid labeling methods.3.direct labeling of oligonucleotid labeling a novel, non-nucleotid, 2-aminobutyl-1, 3-propanediol"Nucleic Acids Res.20(23): 6253-6259, U.S. Pat. No. 5,401,837 issued to Nelson at 28.3.1995 entitled "Method for labeling the 3' end of a synthetic oligonucleotide using a unique Multifunctional oligonucleotide-controlled pore glass (MF-CPG) reagent in colloidal phase oligonucleotide synthesis", and U.S. Pat. No. 5,141,813 issued to Nelson at 25.8.1992 entitled "labeled oligonucleotide for colloidal phase oligonucleotide synthesis").

In certain embodiments, modified nucleotides are included in the blocked oligonucleotides described herein. To illustrate, modified nucleotide substitutions are introduced into oligonucleotide sequences, which can, for example, alter the melting temperature of the oligonucleotide as desired. In some embodiments, this can result in greater sensitivity relative to unmodified oligonucleotides, even in the presence of one or more mismatches in sequence between the target nucleic acid and a particular oligonucleotide. Exemplary modified nucleotides that may be substituted or added to the oligonucleotide include, for example, C5-ethyl-dC, C5-methyl-dC, C5-ethyl-dU, 2, 6-diaminopurine, C5-propynyl-dC, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudo-dU, nitropyrrole, nitroindole, 2 '-0-methyl Ribo-U, 2' -o-methyl Ribo-C, 8-aza-dA, 8-aza-dG, 7-deaza-dA, 7-deaza-dG, N4-ethyl-dC and N6-methyl-dA. To further illustrate, other examples of modified oligonucleotides include those having one or more LNATM monomers. Nucleotide analogs such as these are also described, for example, IN U.S. patent No.6,639,059 entitled "SYNTHESIS OF [2.2.1] bicycclo nuclcoisides" issued to Kochkine et al at 28.10.2003, U.S. patent No.6,303,315 entitled "one SAMPLE PREPARATION AND DETECTION OF nucleccac IN COMPLEX BIOLOGICAL SAMPLEs" issued at 16.10.2001, AND U.S. patent application publication No. 2003/0092905 entitled "SYNTHESIS OF [2.2.1] bicycclo nuclcoisides" issued at 15.5.2003. Oligonucleotides comprising LNATM monomers are commercially available from, for example, Exiqon A/S (Vedbaek, DK). Other oligonucleotide modifications are mentioned herein, including those mentioned in the above definitions.

Nucleic acid incorporation biocatalysts

The blocked oligonucleotides described herein are generally not extendable by at least one nucleic acid incorporating biocatalyst selected from the group consisting of: for example, G46E E678G CS5DNA polymerase, G46E L329A E678GCS 5DNA polymerase, G46E L329A D640G S671F CS5DNA polymerase, G46EL329A D640G S671F E678G CS5DNA polymerase, G46E E678G CS6DNA polymerase, Δ Z05R polymerase, E615G Taq DNA polymerase, Thermus flavus polymerase, TMA-25 polymerase, E678G TMA-25 polymerase, TMA-30 polymerase, E678G TMA-30 polymerase, Tth DNA polymerase, Thermus SPS-17 polymerase, E615G Taq polymerase, Thermus Z05R polymerase, T7DNA polymerase, Kornberg DNA polymerase I, Klinenosol DNA polymerase, Taq DNA polymerase, Micrococcus DNA polymerase, alpha DNA polymerase, reverse transcriptaseAMV reverse transcriptase, M-MuLV reverse transcriptase, DNA polymerase, RNA polymerase, Escherichia coli RNA polymerase, SP6 RNA polymerase, T3 RNA polymerase, T4 DNA polymerase, T7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, ribonucleotide-incorporating DNA polymerase, and the like. Certain such nucleotide-incorporating biocatalyst sequences are publicly available from a variety of sources, including, for exampleAnd the like. To further illustrate aspects of the invention, FIG. 11 schematically depicts a polymerase bound to a template nucleic acid and bound to a primer nucleic acid containing an incorporated cytosine tetraphosphate nucleotide.

One polymerase that can incorporate, but is generally not capable of extending, a 2 '-terminator nucleotide of the invention lacks the F to Y mutation in helix O or conversely lacks the mutation to increase 3' -deoxynucleotide incorporation by the enzyme. Optionally, the enzyme comprises 3 '-5' exonuclease activity and/or is a thermostable enzyme. The enzymes are generally derived from organisms such as Thermus antarikinii, Thermus aquaticus (Thermus aquaticus), Thermus caldophilus, Thermus chalarphilus, Thermus filiformis, Thermus flavus (Thermus flavus), Thermus igniterae, Thermus lactis, Thermus ositima, Thermus rhodobacter (Thermus ruber), Thermus rubens, Thermus scodottus, Thermus farinosus (Thermus silvarlus), Thermus canus (Thermus spec) Z05, Thermus sp 17, Thermus thermophilus (Thermus thermophilus), Thermus maritima (Thermotoga mariticum), Thermus thermophilus (Thermophilus), Thermophilus Bacillus thermophilus (Thermophilus), Bacillus stearothermophilus, et al, Thermoascus faecalis, Thermophilus faecalia lipocalinax, etc.

In some embodiments, the enzyme is modified. Exemplary modified enzymes include, for example, G46EE678G CS5DNA polymerase, G46E L329A E678G CS5DNA polymerase, G46EL329A D640G S671F CS5DNA polymerase, G46E L329A D640G S671F E678GCS 5DNA polymerase, G46E E678G CS6DNA polymerase, E615G Taq DNA polymerase, and the like. Generally, these modified enzymes have an increased ability to incorporate 2' -terminator nucleotides relative to unmodified enzymes. These modified enzymes generally comprise mutations that enhance incorporation of ribonucleotides, enhance incorporation of 2 '-modified analogs of ribonucleotides (e.g., 2' -terminator nucleotides), and/or reduce or eliminate 5 '-3' exonuclease activity (e.g., relative to enzymes lacking one or more of these mutations). Additional details regarding useful biocatalysts are also provided in, for example, U.S. patent application No.60/852882 to Bauer et al, entitled "MUTANDNAPOLYMERASES AND RELATED METHOD", U.S. patent No. 5,939,292 to Gelfand et al, entitled "THERMOSTABLENDNAPOLYMERASES HAVISION REDUCED DISCRIMINATION AINENST RIBO-NTPS", U.S. patent No. 4,889,818 to Gelfand et al, entitled "PURIFIED THERMOSTAMERASENZYME", U.S. patent No. 39CACCARA 34 to Gelfand et al, entitled "TRANSFIED THERMOSTAMERASENZYME", U.S. patent No. 5 CLETRANS TRANS VANE, U.S. 5 TRANS VAN, No. 5, MARE TRANS 5, No. 2, No. TRANS, U.S. patent No. 5,466,591 entitled "5 'TO 3' EXONUCLEASEMUTATIONS OF THERMOSTABLE DNACERASES" issued on 14.11.1995, U.S. patent No. 5,618,711 entitled "RECOMBINANT EXPRESSION VECTORS AND PURIFICATION METHODS FOR THERMOPHOBIC DNACERASE" issued on 8.4.1997, U.S. patent No. 5,624,833 entitled "RECONFANCE EXPRESSION AND PURIFICATION METHYL TRANSPORUS DNACERASE" issued on 29.4.1997, U.S. patent No. 5,674,738 entitled "PUMRIFIED THERMOSTABLE ACPOLYMERA POLYMERA FRETHERAMOMAGA 1997", U.S. patent No. 16 entitled "TRANSPORTION PATION No. 3932 issued on 7.10.7.4.4.4.4.4.4.1997, U.S. patent No. 2 entitled" TRANSPORTION PATENT 1998, "U.S. US patent No. 16,1998 issued on 3,1998, entitled "HIGH TEMPERATURE REVERSE TRANSCRIPTION USE MULTIDNAPOLYSES" and U.S. patent application No. 10/401,403 filed 26/3/2003.

Preparation of modified enzymes having, for example, enhanced efficiency of incorporation of 2' -terminator nucleotides or other desirable properties can be achieved by a variety of methods including, for example, site-directed mutagenesis, chemical modification, and the like. More specifically, site-directed mutagenesis is typically achieved by site-specific primer-directed mutagenesis. This technique is generally performed using synthetic oligonucleotide primers complementary to the single-stranded phage DNA to be mutagenized, except that limited mismatches represent the desired mutation. Briefly, synthetic oligonucleotides are used as primers to direct the synthesis of complementary strands to a plasmid or phage, and the resulting double-stranded DNA is transformed into a phage-supporting host bacterium. The resulting bacteria are detected by, for example, DNA sequence analysis or probe hybridization to identify those plaques carrying the desired mutant gene sequence. To further illustrate, a number of other methods of modifying nucleic acids, such as "recombinant PCR" methods, can also be used.

In practicing aspects of the invention (e.g., preparing modified enzymes, performing sequencing reactions, etc.), many conventional techniques of molecular biology and recombinant DNA are optionally used. These techniques are well known and explained in, for example,Current Protocols in Molecular Biologyvolumes I, II, and III, 1997(f.m. ausubel ed.); sambrook et al, 2001,Molecular Cloning：A Laboratory Manualthird Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; the combination of Berger and Kimmel,Guide to Molecular Cloning Techniques，Methods in Enzymology volume 152 Academic Press，Inc.，SanDiego，CA(Berger)，DNA Cloning：A Practical Approachvolumes I and II, 1985(d.n. glover ed.);Oligonucleotide Synthesis，1984(M.L.Gait ed.)；Nucleic Acid Hybridization1985, (Hames and Higgins);Transcription and Translation1984 (compiled by Hames and Higgins);Animal Cell Culture，1986(R.I.Freshney ed.)；Imm ohilized Cells and Enzymes，1986(IRL Press)；Perbal，1984，A Practical Guide to Molecular Cloning；the series，Methods in Enzymology(Academic Press，Inc.)；Gene Transfer Vectors for Mammalian Cells1987 (compiled by j.h. miller and m.p.calos, Cold Spring harbor laboratory); andMethods in Enzymologyvol.154 and Vol.155 (compiled by Wu and Grossman, and Wu, respectively).

Commercial Process

In other representative embodiments, the invention provides methods of doing business involving the blocked oligonucleotides and polynucleotides described herein. For example, FIG. 24 is a block diagram illustrating certain steps performed in a business method in accordance with one embodiment of the present invention. As shown in step 2400, the method includes receiving an order from a customer for blocked oligonucleotides and polynucleotides described herein and/or instructions for preparing the list of oligonucleotides or polynucleotides. In addition, the method includes providing the oligonucleotide or polynucleotide and/or instructions to the customer in response to the order (step 2402). In some embodiments, for example, the business entity receives the order by visiting in person with a customer or an agent thereof, by mailing or other delivery service (e.g., regular courier), by telephone contact, by email contact or other electronic medium, or any other suitable method. In certain embodiments, the ordered blocked oligonucleotides or polynucleotides and/or instructions are contained in a kit as described herein. Moreover, the blocked oligonucleotide or polynucleotide and/or instructions are provided to the customer (e.g., in exchange for some form of consideration) by any suitable method, including by the customer or an agent thereof visiting himself, by mail, or other delivery service (e.g., a conventional courier).

VIII. kit

The invention also provides kits containing instructions for preparing the blocked oligonucleotides and polynucleotides described herein and/or one or more blocked oligonucleotides and polynucleotides. As described herein, the blocked oligonucleotides and polynucleotides of the invention are generally useful as primers or probes in a variety of nucleic acid technologies. Thus, in certain embodiments, the kit contains, in addition to the blocked oligonucleotides and polynucleotides, other reagents (e.g., buffers, enzymes, etc.) and instructions for performing a particular application (e.g., real-time PCR, PAP-based methods, etc.). In addition, the kits will typically contain containers for packaging the reagents, instructions, and other kit components.

IX. example

It should be understood that the following examples are for illustrative purposes only and are not intended to limit the scope of the claimed invention.

Example 1: regiospecific synthesis of uridine tetraphosphate

FIG. 12 schematically depicts a regiospecific synthetic pathway for uridine tetraphosphate according to an embodiment of the present invention. Note that the numbers in parentheses in this example refer to the compounds shown in FIG. 12.

Synthesis of 5 ' -O-DMT-3 ' -O-TBDMS uridine 2 ' -O- (dicyanoethyl) phosphite [2 ]:

reacting a compound [ 1]](Chemgenes commercial number ANP-4845, 0.680g, 0.790mmol) was placed in acetonitrile (Aldrich, anhydrous, 10 mL). 1-H-tetrazole (Aldrich, 0.211g, 3.01mmol) was added all at once to the above solution, and then to the solution was added againTo this was added 3-hydroxypropionitrile (Aldrich, 0.109mL, 1.58 mmol). The resulting solution was stirred at room temperature for 1 hour under argon atmosphere. The solvent was removed on a rotary evaporator. The residue was taken up in EtOAc (50mL) and washed with saturated NaHCO₃The resulting solution was washed with aqueous solution (2 × 20 mL). Separating the organic layer with MgSO₄And (5) drying. Filtration gave the crude product, which was then evaporated. Before loading the product mixture, CH is used₂Cl₂2% Et in (200mL)₃The Biotage 40S flash cartridge (flash cartridge) was pretreated by N solution elution. Product mixture at very low CH₂Cl₂The solution in (a) was loaded onto a Biotage column. The product was purified by elution with a separate gradient (applied gradient) consisting of 2% Et₃N/98％ CH₂Cl₂(200mL), 0.5% methanol/2% Et₃N/97.5％ CH₂Cl₂(200mL), 1% methanol/2% Et₃N/97％ CH₂Cl₂(200 mL). The purified product was eluted in 0.5-1.0% methanol solvent strength fractions (strength fractions). The fractions containing the purified product were mixed and the solvent was removed on a rotary evaporator. The purified product [2] was obtained in the manner described above]As a white foam (0.510g, 78% yield).

Synthesis of 5 ' -OH-3 ' -O-TBDMS uridine 2 ' -O- (biscyanoethyl) phosphate [4 ]:

reacting the compound [2] at room temperature](0.335g, 0.403mmol) was dissolved in THF (Aldrich, anhydrous, 6.7 mL). Stirring and mixing I₂In pyridine/THF/H₂A solution in O (0.02M, 2.2: 6.8: 1; Glen Research, 24mL, 0.48mmol) was added to the above mixture. The resulting solution was stirred at room temperature for 20 minutes. Aqueous sodium bisulfite solution (1g in 3mL H) was added dropwise₂In O) until in solution I₂The color of (1) is eliminated (queued). The volatile solvent was removed on a rotary evaporator. The solution was diluted with EtOAc to a total volume of 100 mL. With saturated NaHCO₃The solution was carefully washed with aqueous solution (50 mL). The organic layer was separated and MgSO₄And (5) drying. The solution was filtered and the solvent was removed on a rotary evaporator to obtain compound [3 ]]. Crude product [3]Is placed in CH₂Cl₂(8mL), the mixture was stirred. The resulting solution was cooled to-30 deg.f. Trichloroacetic acid (Fisher, 0.487g, 2.98mmol) in CH₂Cl₂(4mL) was added to the cooled and stirred nucleoside solution. The reddish-brown color characteristic of the trityl cation immediately appeared. After adding trichloroacetic acid, stirring was continued at-30 ℃ for 20 minutes. Methanol (1.5mL) was added to the above solution, and the resulting solution was transferred to a separatory funnel. By CH₂Cl₂The solution was diluted (75 mL). The resulting solution was saturated NaHCO₃Aqueous (2 × 30mL) washes. Separating the organic layer with Na₂SO₄And (5) drying. The solution was filtered and the solvent was removed on a rotary evaporator. The crude product was purified by flash column chromatography on silica gel using a 40S Biotage cartridge. The product is in a very small amount of CH₂Cl₂The solution was loaded onto the top of a Biotage column. The product was eluted with a step gradient: EtOAc (200mL), 1% MeOH in EtOAc (200mL), 2% MeOH in EtOAc (200mL), 3% MeOH in EtOAc (200mL), 4% MeOH in EtOAc (200mL), 5% MeOH in EtOAc (200mL) and 10% MeOH in EtOAc (200mL) eluted the product with a 10% MeOH solvent strength component. The fractions containing the purified product were mixed and the solvent was removed on a rotary evaporator. The purified product [4] was obtained in the manner described above]As a white foam (0.145g, 66% yield).

Converting the compound [4] to its corresponding triphosphate [7 ]:

co-evaporation with pyridine (3X0.2mL) to dry 5 ' -OH-3 ' -O-TBDMS uridine 2 ' -O- (dicyanoethyl) phosphate (Compound [4]]0.0335g, 0.0615 mmol). The resulting material was placed in pyridine (Aldrich, anhydrous, 70. mu.L) and DMF (Aldrich, anhydrous, 180. mu.L). A solution of salicylchlorophosphinic acid (Salicylphosphochlorydite) in DMF (0.5M, 137. mu.L, 0.0677mmol) was added to the stirred solution. The resulting reaction mixture was stirred at room temperature for 20 minutes. Add tri-n. butylamine (Aldrich, 38 μ L, 0.160mmol) followed by a solution of tetrabutylammonium pyrophosphate in DMF (0.5M, 185 μ L, 0.0925 mmol). The resulting reaction mixture was stirred at room temperature for 20 minutes. Plus with addition of I₂pyridine/H₂O/THF (0.02MGlen Research, 3.6mL, 0.072mmol) and the resulting reaction mixture stirred at room temperature for an additional 20 minutes. Sodium bisulfite solution (1g NaHSO) was added dropwise₃3mL of water) quenched the excess iodine until the characteristic color of iodine disappeared. The resulting solution was allowed to stand at room temperature overnight. Note that standing at-20 ℃ for 72 hours converted the cyclic triphosphate to linear triphosphate. At this time, no cyclotriphosphate was detected by flow injection Mass Spectrometry (MS). The linear triphosphates produced were subjected to reverse phase HPLC (column: Zorbax SB-C18, 21.2mm x25cm, solvent A: 0.1M TEAA, 2.5% CH)₃CN, pH 7.0; solvent B: CH (CH)₃CN, flow rate: 10.0 mL/min, gradient: t ═ 0 min s, 100% a; t 15 min, 50% a/50% B; t 20 min, 100% Bt 25 min, 100% B; t 25.01 min, 100% a; t 30 min, 100% a, residence time 20.9 min).

3' -TBDMS-uridine triphosphate [8] synthesis:

2' -O-bis (O-cyanoethyl) uridine phosphate [7]](9mg, 0.0117mmol) and MeOH (3X5mL), CH₂Cl₂(3x5mL), anhydrous CH₃CN (1x5mL) was co-evaporated in sequence. This material was then dissolved in CH₃CN (Aldrich, anhydrous, 2.25 mL). 1, 8-bicyclic diazo [5.4.0 ]]Undecyl-7 ene (DBU, Aldrich, 175. mu.L, 1.17mmol) and chlorotrimethylsilane (Aldrich, 59. mu.L, 0.468mmol) were added to the stirred solution. The reaction mixture was stirred at room temperature for 2 hours. Water (1mL) was added and the volatiles were removed on a rotary evaporator. The product obtained was purified by reverse phase HPLC. (column: Zorbax SB-C1821.2mmx25cm, solvent A: 0.1M TEAA, 2.5% CH₃CN, pH 7.0; solvent BCH₃CN, flow rate: 10.0 mL/min, gradient: t-0 min, 100% a; t 15 min, 50% a/50% B; t-20 min, 100% B; t 25 min, 100% B; t 25.01 min, 100% At 30 min, 100% a, residence time: 13.6 minutes). Compound [8] thus obtained]Quantification was performed by UV after lyophilization (8 mg). Molar extinction coefficient (. epsilon.) of uridine_max) Is 10 (mM)^-1cm^-1) Maximum absorption peak (. lamda.)_max) At 262 nm.

Synthesis of uridine triphosphate [9 ]:

3' -O-TBDMS-uridine tetraphosphate [8]](2.55mg, 0.00376mmol) in CH₃CN (Aldrich anhydrous, 160. mu.L). To the above solution was added tetrabutylammonium fluoride in THF (1.0M, Aldrich, 113. mu.L, 0.113mmol) and HOAc (ice, Aldrich, 2.2. mu.L, 0.0376 mmol). The resulting reaction mixture was stirred at room temperature for 21 hours. HPLC analysis of the reaction at this point showed no residual silyl ether. Volatiles were removed using a rotary evaporator. The product mixture was resuspended in water and the product purified by reverse phase HPLC. (column: Zorbax SB-C18, 9.4mmx25cm, solvent A: 0.1M TEAA, 2.5% CH₃CN, pH 7.0; solvent B: CH (CH)₃CN, flow rate: gradient 4.0 mL/min: t-0 min, 100% a; t 15 min, 75% a/25% B; t 15.01 min, 100% B; t-20 min, 100% a; t-27 min, 100% a, residence time: 7.05 minutes). The purified product was lyophilized. The resulting material was resuspended and lyophilized a total of 5 times to ensure complete removal of the TEAA salt. The resulting material was quantified with UV (2.00mg, 94% yield) prior to final lyophilization. Compound [9] obtained in the above manner]Is a white solid.

Example 2: synthesis of adenosine tetraphosphate

This example illustrates the synthesis of adenosine tetraphosphate according to one of the synthetic reactions of the present invention. As schematically shown in FIG. 13, this synthetic reaction produced a mixture of adenosine 5 '-triphosphate-3' -monophosphate and adenosine 5 '-triphosphate-2' -monophosphate. In this synthesis, ATP and trisodium trimetaphosphate (NaPO) are added at room temperature₃)₃Reaction in 1N KOH. The reaction was carried out for 18 hours. The molar ratio of adenosine 5 '-triphosphate-3' -monophosphate to adenosine 5 '-triphosphate-2' -monophosphate is about 50: 50.

FIGS. 14A-C are HPLC profiles (abscissa-retention time (min); ordinate-260 nm Absorbance Units (AU)), showing detection of various adenine nucleotides. The profile was generated after nucleotide separation by ion-coupled RP-HPLC chromatography. In particular, in symmetrytryShield^TMAnalysis and purification was performed on reverse phase columns (Waters Corporation, Milford, Mass.) and the buffer was TEAA-acetonitrile. FIG. 14A shows HPLC analysis of adenosine tetraphosphate, while FIGS. 14B and C illustrate HPLC analysis of purified adenosine tetraphosphate fractions (retention times: 4.166 and 4.52 minutes, respectively). Independent NMR analysis (³¹p NMR; 2D proton-phosphorus NMR) (described further below) showed that the peak eluting at 4.33 minutes corresponded to 2' -PO₄ATP, peak eluted at 4.65 min corresponding to 3' -PO₄ATP (see, FIG. 14A).

NMR analysis

Adenosine tetraphosphate corresponding to peak eluted at 4.33 min in the above HPLC analysis¹H and³¹the chemical shifts for p are shown in Table 1. Assignment of proton chemical shifts (assignment) using COSY spectra, in fact, proton H-1' is easily identified because there should be only one proton coupled and it should be the furthest low magnetic field (furthst downfield) of the ribose protons.

TABLE 1

Atom(s)	1H shift (ppm), Multiplicity (multiplicities), fragmentation (splits)	31P shift (ppm), multiplicity, P-P cleavage
			2	8.50，s	-
8	8.27，s	-
			1′	6.27，d，5.4Hz	-
2′	5.05，d of t，8.8，5.5Hz	-
			3′	4.67，d of d，5.4，4.1Hz	-
4′	4.41，m	-
			5′	4.26，d of d，5.2.3.3Hz	-

P2′	-	0.87，s
			Pα	-	-10.69，d，19.5Hz
Pβ	-	-22.44，t，19.5Hz
			Pγ	-	-9.92，d，19.5Hz

Also obtained with or without proton uncoupling³¹P spectrum.³¹Four phosphate peaks were observed in the P spectrum. The three peaks at-22.44, -10.69, and-9.92 ppm show³¹P-³¹p-coupling, a triphosphate group at the 5' position of the ribose ring. The fourth peak at 0.87ppm did not appear³¹p-³¹p-coupling, belonging to the monophosphates. However, monophosphates in two adenosine tetraphosphates (i.e., peaks at 4.33 and 4.65 minutes)³¹The p chemical shifts are very similar, 0.87 and 1.08ppm respectively, and therefore cannot be used to determine the position of the phosphate group. Furthermore, the line widths of the mono-and triphosphate peaks are very large (broad) and are therefore not coupled without proton uncoupling³¹Failure to resolve into p spectrum³¹p ¹H coupling.

Comparison of the H-2 ' and H-3 ' chemical shifts in adenosine 5 ' -monophosphate, adenosine 2 ' -monophosphate, and adenosine tetraphosphate (peak at 4.33 min) showed that the monophosphate bound to the C-2 ' of adenosine tetraphosphate corresponding to the peak at 4.33 min (see Table II). In addition, H-2' (confirmation) which has two adjacent protons and is therefore expected to be a doublet or triplet, is a doublet. The other coupling at 8.8Hz is exactly a triple bond³¹p-¹H-coupled value (magnitude). Based on these results, the structure of adenosine tetraphosphate of the peak (corresponding) at 4.33 minutes, that is, corresponding to 2' -PO, was determined₄-ATP。2’-PO₄Structural representation of-ATPFigure 13 is schematically shown.

TABLE II

Molecule	H-2′(ppm)	H-3′(ppm)
			5' -adenosine phosphate	4.75	4.51
2' -monophosphate adenosine	5.19	4.57
			Adenosine tetraphosphate (4.33 minutes)	5.05	4.67

The 4.65 minute peak corresponds to a mixture of nucleotides, showing a seemingly large amount of triethylamine salt. Table III shows the nucleotide sequence¹H and³¹p chemical shifts and measured splits (splits) by¹H and³¹and (5) detecting a p spectrum. As with the 4.33 minute adenosine tetraphosphate peak, assignment of proton chemical shifts using COSY spectra, in fact, proton H-1' is readily identified because there should be only one proton coupled and should be the lowest magnetic field farthest among the ribose protons. Due to and above adenosine tetraphosphate peak for 4.33 minFor the same reason, without coupling or decoupling³¹P spectrum.

TABLE III

Atom(s)	1H shift (ppm), multiplicity, fragmentation	31P shift (ppm), multiplicity, P-P cleavage
			2	8.56，s	-
8	8.27，s	-
			1′	6.18，d 6.7Hz	-
2′	～4.8，m	-
			3′	～4.8，m	-
4′	4.60，p，～2.8	-
			5a′	4.28，d of d of d，12.0，5.2，2.8	-
5b′	4.23，d of d of d，12.0，4.4，2.6	-
			P3′	-	1.08，s
Pα	-	-10.70，d，19.0Hz
			Pβ	-	-22.42，t，19.0Hz
Pγ	-	-9.92，d，19.0Hz

For the adenosine tetraphosphate sample corresponding to the 4.65 minute peak, the chemical shifts for both H-2 'and H-3' were about 4.8 ppm. Thus, H-2 ' moves backward, high magnetic field (moved back up), approaching the chemical shift of H-2 ' in adenosine 5 ' -phosphate, 4.75ppm (see, Table II). Relative to the chemical shifts (see Table II) of the two adenosine 5 '-phosphate (4.51ppm) and adenosine tetraphosphate (peak at 4.33 min) (4.67ppm), H-3' moves towards a low magnetic field. Based on these phenomena, the structure of adenosine tetraphosphate of a peak (corresponding) at 4.65 minutes, i.e., corresponding to 3' -PO, was determined₄-ATP。3’-PO₄The structure of ATP is schematically shown in FIG. 13.

Example 3: synthesis of tamra labeled 2 '-monophosphate-uridine triphosphate and 3' -monophosphate-uridine triphosphate

This example describes the synthetic pathways of labeled 2 '-monophosphate-uridine triphosphate and 3' -monophosphate-uridine triphosphate according to one embodiment of the present invention. For further explanation, FIG. 15 schematically shows some of the steps of the pathway described in this example. Note that, the numerals in parentheses in this example represent the compounds shown in fig. 15.

Compound [3]Dissolved in H₂To O (300. mu.L), 100. mu.L of the solution was added to the flask. The solution in the flask was diluted with 1.0mL of 1N KOH. Sodium trimetaphosphate (50mg) was added to the solution, and the solution was stirred at room temperature for 1 hour. 50mg of sodium trimetaphosphate was added thereto, and stirring was continued for 2 hours. Then 50mg of sodium trimetaphosphate were added thereto, and stirring was continued at room temperature overnight. The next day, 80. mu.L of glacial acetic acid was added to a pH of about 7.0. The resulting reaction mixture was purified by RP-HPLC. First, the nucleoside-containing reaction mixture is partially separated from the salt. After lyophilization, the tetraphosphate salt is separated from the starting material. FIG. 16 is a chromatogram (abscissa-retention time (min); ordinate-Absorbance Units (AU) at 290 nm) showing the detection of these tetraphosphates. Specifically, the peaks eluting at 15.9 and 16.1 minutes correspond to the tetraphosphate salt, and the peak eluting at 16.8 minutes corresponds to the starting material.

7mg of the isomer mixture of tetraphosphate (compounds [4] and [5]) was placed in 200. mu.L of trifluoroacetic acid (TFA) at room temperature. The resulting solution was stirred for 30 minutes. The solution was then cooled in liquid nitrogen and lyophilized to remove TFA. The obtained substance was then reacted with carboxytetramethylrhodamine succinimidyl ester (TAMRA-SE) without further operation.

Stock solutions of TAMRA-SE were prepared by dissolving 5mg of TAMRA-SE in 350. mu.L of Dimethylformamide (DMF). In addition to this, the present invention is,at 100 μ L H₂Dissolving 6mg of tetraphosphoric acid compound in O to prepare a stock solution of tetraphosphoric acid compound (containing compound [6 ]]And [7]])。

Transfer of TAMRA-SE stock to a stock containing 175. mu. L H₂O in a conical flask. In addition, 1.875. mu.L of a marker buffer (0.1M sodium tetraborate) and a stock solution of tetraphosphate compound were added to the mixture. The resulting mixture was stirred overnight at room temperature in the dark (the flask was covered with aluminum foil). The next day, 100. mu.L of the reaction mixture was subjected to HPLC separation, showing that two peaks eluted at about 17 minutes, which was considered as a labeled tetraphosphate compound isomer (compound [8]]And [9]]). The fractions corresponding to these peaks were separated from the remaining reaction mixture by RP-HPLC. The collected sample was lyophilized and concentrated, and flow injection analysis-Mass Spectrometry (FIA-MS) showed 1027.8 ion (peak) corresponding to expected (M-H)^-The ion mass deviates from one mass unit. The remaining reaction mixture was stirred at room temperature overnight.

We believe that since the fluorescent label is positively charged, removal of one proton (M-H) will produce a molecule that is generally neutral. Thus, the molecules produced (M-2H) should be mass: the charge ratio was-1. This probably explains the molecular ions observed above.

Other exemplary synthetic routes to other labeled tetraphosphate compounds are illustrated in FIGS. 17-19. In particular, FIG. 17 schematically illustrates certain steps in the synthetic pathway of ROX-labeled cytidine tetraphosphate. FIG. 18 schematically depicts certain steps in the R6G-labeled adenosine tetraphosphate synthesis pathway. FIG. 19 schematically shows certain steps in the R110-labeled guanosine tetraphosphate synthesis pathway.

Example IV: blocking extension of primer nucleic acids with 2' -terminator nucleotides

This example shows a comparison of two primer nucleic acid extension reactions. FIG. 20 shows the sequences of the template and primer nucleic acids used in these analyses, corresponding to the sequence names NJS115 and NJS01, respectively^＊. FIGS. 20A and B are electropherograms illustrating that in reactions that do not include terminator nucleotidesIncorporation of adenine and guanine residues into NJS01^＊In (1). In contrast, the electropherograms of FIGS. 20C and D illustrate that NJS01 is caused by incorporation of the adenosine tetraphosphate terminator nucleotides of the present invention^＊Cannot be extended.

Example V: automated cycle sequencing with modified thermostable DNA polymerases and fluorescent primers

This example illustrates the use of the 2' -terminator nucleotides of the present invention for automated dye primer cycle DNA sequencing. Specifically, the M13mp18 DNA template was sequenced with 2 '-monophosphate 5' -ribonucleoside triphosphate.

The cycle sequencing reaction was performed with G46E E678G CS5DNA polymerase (mentioned above) modified for incorporation of ribonucleotide analogs, dye primers, and 2 '-monophosphate 5' -ribonucleoside triphosphate analogs. The reaction consisted of the following components: 50mM zwitterionic buffer (Tricine) pH 8.5; 40mM KOAc; 4mM Mg (OAc)₂(ii) a dATP, dCTP, dTTP, each 100. mu.M; 150 mu Mc7 dGTP; 0.5 units/. mu. l G46E E678G CS5DNA polymerase; 1.0 units/. mu.l of rTth thermostable pyrophosphatase; and 20 ng/. mu. l M13mp18 template. Each base was reacted independently for four times. The reaction of each base contains the following reagents in addition to the above components:

adenosine reaction (10. mu.l)

3.5. mu.M 2 '-monophosphate 5' -adenosine triphosphate

0.1 μ M FR686NHEX primer

Cytidine reaction (10. mu.l)

7.5. mu.M 2 '-monophosphate 5' -triphosphate cytidine

0.1. mu.M FR686NFAM primer

Guanosine reaction (20. mu.l)

5 μ M guanosine 2 '-monophosphate 5' -triphosphate

0.1. mu.M FR686NTAMRA primer

Uridine reaction (20. mu.l)

10 μ M uridine 2 '-monophosphate 5' -triphosphate

0.1. mu.M FR686NROX primer

In adenosine reactions, adenosine-5 '-monophosphate is about 95% pure (i.e., about 5% is adenosine-5' -monophosphate). In the cytidine reaction, cytidine 5 '-triphosphate 2' -monophosphate and cytidine 5 '-triphosphate 3' -monophosphate constitute 50/50 mixtures. Guanosine 2 '-monophosphate 5' -triphosphate is about 94% pure in the guanosine reaction (i.e., about 6% was guanosine 3 '-monophosphate 5' -triphosphate). In the uridine reaction, the purity of uridine 2 '-monophosphate 5' -triphosphate is 100%.

The oligonucleotide primer sequences were as follows:

FR686NFAM FCGCCAGGGTTTTCCCAGTEA

2 '-amino (ribose) C F ═ 5' FAM ABD

FR686NHEX ICGCCAGGGTTTTCCCAGTEA

2 '-amino (ribose) C I ═ 5' HEX ABD

FR686NROX JCGCCAGGGTTTTCCCAGTEA

2' -amino (ribose) C J ═ 6-ROX

FR686NTAMRA LCGCCAGGGTTTTCCCAGTEA

2' -amino (ribose) C L ═ C6-amino TAMRA

These four reactions were each placed in a Perkin-ElmerIn the PCR system 9600 thermal cycler, the reaction conditions were: 45 seconds at 95 ℃; 20 cycles: 15 seconds at 95 ℃, 15 seconds at 55 ℃ and 90 seconds at 70 ℃; 20 cycles: 95 ℃ for 15 seconds, 70 ℃ for 90 seconds. The four reactions were pooled (pool) and 144. mu.l was added100% ethanol and 6. mu.l 3M NaOAc (pH 5.2) were precipitated for 15 minutes at 4 ℃. The pooled reaction was centrifuged at 4 ℃ for 15 minutes in a microcentrifuge to precipitate the DNA, and the supernatant was removed. The centrifuged precipitate was washed with 350. mu.l of cold 70% ethanol, centrifuged at 4 ℃ for 5 minutes in a microcentrifuge, the supernatant was removed and the DNA precipitate was dried. The precipitated DNA was heated at 90 ℃ for 3 minutes in 10. mu.l of Hi-Di formamide (Applied Biosystems, Foster City, CA, part #4311320), and placed on ice. 2 μ l of each sample was loaded onto pre-electrophosphoresced 48cm 4.25% acylamide: bis (29: 1), 6M Urea gel on ABI PRISM^TM377 DNA sequencer (Applied Biosystems, Foster City, Calif.) for 7 hours.

Data analysis was performed with sequencing analysis software 3.4.1(Applied Biosystems, Foster City, CA) where the primer file used (primer file) was DP 4% Ac { KS } (semi-adapted basedial version 3.3.1b2(semiadaptive basedial version 3.3.1b2)), and a matrix file (matrix file) specific for the dye primers used above, constructed according to the Applied Biosystems usage manual (No. # 903436). The automatic dot-naming (baselisting) step performed by the analysis software has 100% accuracy for bases +18- +739 of the sequencing primer when compared to the M13mp18 reference sequence. Figure 21 provides a data map of this sequencing analysis.

Example VI: cyclic DNA primer extension using modified thermostable DNA polymerases and dye-labeled 2 '-monophosphate 5' -ribonucleoside triphosphate

A thermocycling primer extension reaction was performed with modified RNA polymerase for incorporation of the ribonucleoside analogue G46E E678G CS5, unlabeled primer and TAMRA dye-labeled uridine 2 '-monophosphate 5' -triphosphate. The composition of the 20 μ l reaction was: 50mM zwitterionic buffer pH 7.5; 25mM KOAc; 2.5mM Mg (OAc)₂(ii) a 100. mu.M each of dATP, dCTP, and dTTP; 150 μ M dITP; 0.5 units/. mu.lG 46E E678G CS5DNA polymerase; 1.0 units/. mu.l of rTth thermostable inorganic pyrophosphatase; 5 ng/. mu. l M13mp18 template; 0.15. mu.M primer; and 0.25. mu.M TAMRA-uridine 2 '-phosphate 5' -triphosphate.

Control reactions were performed with AmpliTaq DNA polymerase, FS, unlabeled primer and TAMRA dye-labeled ddTTP. The composition of the 20 μ l reaction was: 50mM Tris pH 9; 2mM MgCl₂(ii) a 100. mu.M each of dATP, dCTP, and dTTP; 150 μ M dITP; 0.5 units/. mu.l AmpliTaq DNA polymerase, FS; 1.0 units/. mu.l of rTth thermostable inorganic pyrophosphatase; 5 ng/. mu. l M13mp18 template; 0.15 μ MFR686N primer; and 0.2. mu.M TAMRA-ddTTP.

FR686N CGCCAGGGTTTTCCCAGTEA

E ═ 2' -amino (ribose) C

The reaction was placed in a Perkin-ElmerIn the PCR system 9700 thermal cycler, the reaction conditions were: 20 seconds at 96 ℃; 25 cycles: 96 ℃ for 10 seconds, 50 ℃ for 5 seconds, 60 ℃ for 4 minutes. After cycling the reaction, unincorporated dye-labeled terminator was removed from the reaction by centrifugation through a Sephadex-G50 column (Sigma, Part No G-50-80) at 700Xg for 2 minutes. The sample was heated at 95 ℃ for 3 minutes and placed on ice. Electrophoresis was carried out on an Applied Biosystems 3100 Genetic Analyzer (Genetic Analyzer) using GeneScan with StdSeq 50-POP 6DefaultModule using a 50cm capillary array and POP6 polymer.

Data were analyzed using Applied Biosystems GeneScan 3.7 fragment analysis software. FIG. 22 shows a fragment map of the T peak 77-273 bases (T peaks 77 to 273 bases from primer FR686N) of primer FR 686N. More specifically, comparison of the fragment patterns generated by G46E E678G CS5DNA polymerase and TAMRA-uridine 2 '-monophosphate 5' -triphosphate (column B) with the fragment patterns generated by control AmpliTaq DNA polymerase, FS and TAMRA-ddTTP (column A) revealed similar peak patterns.

Example VII: synthesis of blocked oligonucleotides

Using standard β -cyanoethyl phosphoramidite chemistry (FIG. 26), in an automated applied biosystems 39Synthesis of 2' -O-PO on 4-synthesizer₃Blocked oligonucleotides (FIG. 25). The solid support used in the synthesis of these oligonucleotides is CPG 3 '-phosphate (available from Glen Research, # 20-2900-41), which facilitates the introduction of phosphate groups at the 3' -end of the oligonucleotide. In the first cycle of synthesis, ribonucleoside-2' -O-phosphoramidites from Chemgenes (FIG. 27, where B is base; adenosine (accession # ANP-5681), cytidine (ANP-5682), guanosine (ANP-5683) and uridine (ANP-5684)) were used for coupling to solid supports. In the second cycle of synthesis and thereafter, standard deoxynucleoside phosphoramidites were used. After synthesis, the oligonucleotides were cleaved from the solid support and deprotected with concentrated ammonium hydroxide at room temperature for 24 to 48 hours. The ammonium hydroxide was then removed by size exclusion chromatography (NAP-10 column; eluting with sterile water). The oligonucleotides were purified by reverse phase HPLC (PRP-1 column, triethylammonium acetate-acetonitrile buffer). The purified oligonucleotide was concentrated and treated with potassium fluoride to remove the silyl protection of the 3 '-hydroxyl group at the 3' -terminus of the oligonucleotide. The oligonucleotides were further purified by RP-HPLC (Xterra SB-18 column). The purity and identity of these oligonucleotides was confirmed by ion exchange HPLC (Dionex, pH8.0 at 60 ℃) and LC-MS analysis.

Example VIII: HIV DNA template titration

PAP-related HIV DNA template titrations were performed in the presence and absence of genomic DNA. FIG. 28 is a photograph of a gel showing detection of PCR products under different reaction conditions used in the assay. This data explains, for example, that improved amplification specificity and sensitivity can be achieved using blocked primers as described herein relative to reactions that do not use those primers.

More specifically, reactions were performed using the ABI 5700 sequence detection system using the following temperature profile:

2 minutes at 50 DEG C

1 minute at 93 DEG C

93 ℃, 15 seconds → 52 ℃,4 minutes x4 cycles

90 ℃, 15 seconds → 55 ℃,4 minutes x56 cycles

The following reaction conditions are common to all reactions:

main mixed component	Concentration of
		Zwitterionic buffers (pH 8.0)	100mM
dATP	200μM
		dCTP	200μM
dGTP	200μM
		dTTP	30μM
dUTP	300μM
		Primer 3 or primer 1	200nM
Primer 4 or primer 2	200nM
		KOAc	110mM
SYBR Green I	0.2X
		NaPPi	225μM
Mg(OAc)2	2.5mM

Tth storage buffer (0.2% Tween)	6％v/v
		GLQDSE CS5DNA polymerase	10nM

Note: "GLQDSE CS5DNA polymerase" refers to G46E L329A Q601R D640GS671F E678G CS5DNA polymerase. Note also that: "Tth storage buffer" includes 0.2% Tween 20, 20mM Tris pH8.0, 0.1mM EDTA, 100mM KCl, 1mM DTT, and 50% v/v glycerol. Diethylpyrocarbonate (DEPC) -treated water adjusted each reaction volume to 50. mu.l.

The different reaction components included the following unblocked primers (see, the reaction denoted "unblocked primer" in FIG. 28):

primer 15 '-TGAGACACCAGGAATTAGATATCAGTACAATGT-3'

Primer 25 '-CTAAATCAGATCCTACATATAAGTCATCCATGT-3'

And the following blocked primers (see, the reaction denoted by "blocked primers" in FIG. 28):

primer 3

5’-TGAGACACCAGGAATTAGATATCAGTACAATGU^＊-3’

Primer 45' -CTAAATCAGATCCTACATATAAGTCATCCATGU^＊-3’

Wherein U is^＊Refers to 2 ' -phospho-U (i.e., contains in the 2 ' position of the phosphate group of 2 ' -terminator nucleotides). The reaction also included (see, the reaction denoted "25 ng genomic DNA" in FIG. 28) or lacked (see, the reaction denoted "clean target" in FIG. 28) 25ng of human genomic DNA added to the mixture. As further shown in FIG. 28, the reactants also include 10⁵、10⁴、10³、10²Or 10¹Individual copies of linearized plasmid DNA comprising the target nucleic acid diluted in 1. mu.l HIV sample diluent (10mM Tris, 0.1mM EDTA, 20. mu.g/mL Poly A (Poly A), and 0.09% NaN3), or 1. mu.l HIV sample diluent in a "Neg" reaction. The indicated primer pairs amplified a 170 base pair product from plasmid DNA.

Example IX: amplification of mutant K-Ras plasmid templates in the context of wild-type K-Ras plasmid templates Board

Amplification of plasmid templates containing various copy numbers of mutant K-Ras was performed in the background of wild-type K-Ras plasmid templates and the blocked and unblocked primers were compared. FIG. 29 shows the threshold cycle (C) observed for the various mutant K-Ras plasmid template copy numbers (x-axis) used in these reactions_T) Value (y-axis). FIG. 29 further illustrates that improved discrimination can be achieved using blocked primers as described herein.

The reactions were performed using the ABI 5700 sequence detection system, using the following temperature profile:

2 minutes at 50 DEG C

1 minute at 93 DEG C

92 ℃, 15 seconds → 65 ℃,2 minutes x60 cycles

The following reaction conditions are common to all reactions:

main mixed component	Concentration of
		Zwitterionic buffers (pH 8.0)	100mM
dATP	200μM

dCTP	200μM
		dGTP	200μM
dTTP	30μM
		dUTP	300μM
Primer 7 or primer 5	200nM
		Primer 8 or primer 6	200nM
SYBR Green I	0.1X
		NaPPi	225μM
Mg(OAc)2	2.5mM
		Ung	2U
Tth storage buffer (0.2% Tween)	6％v/v
		GDSE CS5DNA polymerase	5nM
Linearized wild-type plasmid DNA	106Copy (a) of

Note: "GDSE CS5DNA polymerase" refers to G46E D640S 640G S671F E678G CS5DNA polymerase. In addition, the volume of each reaction was adjusted to 50. mu.l with DEPC-treated water.

The different reaction components included the following unblocked primers (see, the reaction denoted "unblocked" in fig. 29):

primer 55 '-AAACTTGTGGTAGTTGGAGCTC-3'

Primer 65 '-GTTGGATCATATTCGTCCACAA-3'

And the following blocked primers (see, the reaction denoted "blocked" in fig. 29):

primer 75' -AAACTTGTGGTAGTTGGAGCTC^＊-3’

Primer 85' -GTTGGATCATATTCGTCCACAA^＊-3’

Wherein C is^＊Refers to 2' -phosphoric acid-C, and A^＊Refers to 2 ' -phospho-A (i.e., contains in the 2 ' position of the phosphate group of the 2 ' -terminator nucleotides). In addition, 10⁶、10⁵、10⁴、10³、10²、10¹Or 0 copies (NTC reaction) (in FIG. 29, 10e6c, 10e5c, 10e4c, 10e3c, 10e2c, 10e1c and NTC, respectively). The relevant subsequences of the mutant plasmid DNA perfectly matched the blocked and unblocked primer sets. In addition, in 1 u l HIV sample diluent (see above) or in the "NTC" reaction in 1 u l HIV sample diluent (see above) dilution mutation of K-Ras plasmid DNA. In addition, 10⁶A single copy of linearized wild-type K-Ras plasmid DNA was present in all reactions. The C was generated except for the last 3' base (dC) in primers 5 and 7: the wild-type K-Ras plasmid DNA has the same sequence as the mutant plasmid DNA except for the C mismatch. Blocked and unblocked primer pairs produced a 92 base pair amplicon on the mutation linearized plasmid template.

Example X: amplification of K-Ras plasmid template with various enzymes at different concentrations

Amplification of the plasmid template containing K-Ras was performed with various enzymes at different concentrations. FIG. 30 is a graph showing the threshold cycles (C) observed for various enzymes and concentrations (x-axis) used in these reactions_T) Value (y-axis). The reactions were performed using the ABI 5700 sequence detection system, using the following temperature profile:

2 minutes at 50 DEG C

1 minute at 93 DEG C

92 ℃, 15 seconds → 60 ℃,2 minutes x60 cycles

The following reaction conditions are common to all reactions:

main mixed component	Concentration of
		Zwitterionic buffers (pH 8.0)	100mM
dATP	200μM
		dCTP	200μM
dGTP	200μM
		dTTP	30μM
dUTP	300μM
		Primer 9	200nM
Primer 10	200nM
		SYBR Green I	0.1X
NaPPi	225μM
		Mg(OAc)2	2.5mM
Ung	2U
		Tth storage buffer (0.2% Tween)	6％v/v
Linearized K-Ras plasmid DNA	104Copy (a) of

The reaction components included the following blocked primers:

primers 95' -AAACTTGTGGTAGTTGGAGCTGU^＊-3’

Primers 105' -GTTGGATCATATTCGTCCACAA^＊-3’

Wherein U is^＊Refers to 2' -phosphoric acid-U and A^＊Refers to 2 ' -phospho-A (i.e., contains in the 2 ' position of the phosphate group of the 2 ' -terminator nucleotides). The primer pair formed a 92 base pair amplicon on the linearized K-Ras plasmid template. Diethylpyrocarbonate (DEPC) -treated water adjusted each reaction volume to 50. mu.l.

The following table shows the optimized concentration and the optimized KOAc concentration for each polymerase:

polymerase enzyme	Polymerase concentration (nM)	KOAc(mM)
			GLQDSE	5. 10, 15, 20, 30 or 40nM	110
GLDSE	5. 10, 15, 20, 30 or 40nM	25
			GLE	5. 10, 15, 20, 30 or 40nM	25

Note: "GLQDSE" (SEQ ID NO: 22) refers to G46E L329A Q601R D640GS671F E678G CS5DNA polymerase; "GLDSE" (SEQ ID NO: 23) refers to G46EL329A D640G S671F E678G CS5DNA polymerase, and "GLE" refers to G46EL329A E678G CS5DNA polymerase.

Example XI: PAP-related enzyme comparison

This example compares the ability of G46E L329A E678G CS5DNA polymerase with G46EL329A D640S671F E678G CS5DNA polymerase to perform pyrophosphorolysis-activated polymerization ("PAP"). The reaction buffer contained 100mM zwitterionic buffer pH8.0, 0mM (G46E L329A E678G CS5DNA polymerase) or 50mM (G46E L329A D640S671F E678G CS5DNA polymerase) KOAc, 10% v/v glycerol, 0.04U/. mu.l UNG, 4mM Mg (OAc)₂0.2X SYBR Green I, 2.5% v/v enzyme storage buffer (50% v/v glycerol, 100mM KCl, 20mM Tris pH8.0, 0.1mM EDTA, 1mM DTT, 0.5% Tween 20), 0.2mM each of dATP, dCTP and dGTP, and 0.4mM dUTP, and 100. mu.M pyrophosphate. The M13 template (GenBank accession number X02513) and enzyme were cross-titrated. The M13 concentrations used were 0, 10⁴、10⁵And 10⁶Copies/20. mu.l reaction. The enzyme concentrations used were 2.5nM, 5nM, 10nM, 15nM, 20nM, 25nM, 35nM and 50 nM. Reactions were established in triplicate in a 384-well thermocycler using the following cycling parameters: 2 minutes at 50 ℃; 1 minute at 90 ℃; then 46 cycles: 90 ℃ for 15 seconds and 62 ℃ for 60 seconds.

The primer sequences used were 5 '-CGCCTGGTCTGTACACCGTTXA-3' (primer 11) and 5 '-GGAACGAGGGTAGCAACGGCTACE-3' (primer 12), where X ═ 2 '-amino-C and E ═ 2' -PO₄A (i.e., 2' -terminator nucleotide). Each of these primers was added to the reaction mixture at 0.1. mu.M, resulting in a 348bp product from the M13 template. To be useful as a primer, primer 12 must be activated by pyrophosphorolysis removal of the terminal residue.

Fluorescence data is analyzed to determine the elbow value (i.e., c (t) (appearance of fluorescence above baseline)). The C (t) value of G46E L329A E678G CS5DNA polymerase is shown in FIG. 31. The C (t) value of G46E L329A D640G S671F E678G CS5DNA polymerase is shown in FIG. 32.

Example XII: hepatitis C comparing unblocked and blocked Reverse Transcription (RT) primers Reverse Transcription (RT) of viral (HCV) RNA into cDNA

Comparison of the extension of unblocked HCV RT primers and the extension of blocked primers on HCV RNA templates in a reverse transcription reaction. These RT comparisons were performed using various polymerases. To illustrate, the graph of FIG. 33 shows threshold cycle (Ct) values (y-axis) observed for various enzymes (x-axis) used in these reactions, where cDNA was measured using real-time PCR comprising a 5' -nuclease probe.

The following reaction conditions are common to all RT reactions:

RT mixture Components	Concentration of
		Zwitterionic buffer pH8.0	100mM
KOAc	100mM
		DMSO	4％(v/v)
Primer 1 or 2	200nM
		dATP	200μM
dCTP	200μM
		dGTP	200μM
dTTP	30μM
		dUTP	300μM
UNG	0.2 unit
		Mn(OAc)2	1mM
PPi	175uM

The different reaction components included the following 3 '-OH unblocked primer (see, the reaction denoted by "3' OH primer (unblocked)" in FIG. 33):

primer 15 '-GCAAGCACCCTATCAGGCAGTACCACAA-3'

And the following blocked primers (see, the reaction represented by "2' PO4 (blocked)" in FIG. 33):

primer 25' -GCAAGCACCCTATCAGGCAGTACCACAA^＊-3’

Wherein A is^＊Refers to 2 ' -phospho-A or 2 ' -monophosphate-3 ' -hydroxy adenosine nucleotides (i.e., containing the 2 ' position of the phosphate group of 2 ' terminator nucleotides). In addition, the following were compared in cDNA reactionPolymerase conditions (see, fig. 33):

ZO5DNA polymerase (13nM)

GLQDSE CS5DNA polymerase (100nM) in combination with GLQDS CS5DNA polymerase (25nM)

GLQDSE CS5DNA polymerase (50nM) in combination with GLQDS CS5DNA polymerase (50nM

Wherein "GLQDSE CS5DNA polymerase" (SEQ ID NO: 22) refers to G46E L329AQ601R D640G S671F E678G CS5DNA polymerase, "and" GLQDS CS5DNA polymerase "(SEQ ID NO: 24) refers to G46E L329A Q601R D640G S671F CS5DNA polymerase. Diethylpyrocarbonate (DEPC) -treated water adjusted each reaction to 20. mu.l.

The RT reactions were incubated for 60 minutes at 60 ℃ in an ABI 9600 thermal cycler. After RT incubation, RT reactions were diluted 100-fold in DEPC treated water. The presence and quantification of cDNA was confirmed by a real-time HCV PCR reaction based on a 5' nuclease probe, designed to specifically measure the HCV cDNA product of the RT reaction. These reactions were performed using an ABIPrism 7700 sequence detector, using the following temperature conditions:

2 minutes at 50 DEG C

95 ℃ for 15 seconds → 60 ℃ for 1 minute x50 cycles.

Example XIII: bidirectional PAP for BRAF mutation detection

Fig. 34 shows the PCR growth curve for BRAF oncogene amplification generated when bidirectional PAP was performed. The x-axis shows the normalized cumulative fluorescence and the y-axis shows the cycles of PAP PCR amplification. More specifically, when BRAF oncogenes are carried out using 2 '-terminator-blocked primers that overlap at their 3' -terminal nucleotides at precise mutation positions (see, Brose et al (2002)Cancer Res62: 6997-7000) the mutation-specific amplification of the T → A mutation responsible for the codon change of V599E, generated these data. When the wild-type sequence-specific primer is used with the wild-type target or the mutant targetIn the reaction, only wild-type target was detected. In contrast, when a primer specific for the mutated sequence reacts with either the wild-type target or the mutated target, only the mutated target is detected.

The following reaction conditions are common to all RT reactions:

components	Concentration of
		Zwitterionic buffer pH8.0	100mM
KOAc	100mM
		Glycerol	3.5％v/v
Primers F5W or F5M	200nM
		Primer R5W or R5M	200nM
dATP	200μM
		dCTP	200μM
dGTP	200μM
		dTTP	30μM
dUTP	300μM
		UNG	1 unit of
PPi	175uM

GLQDSE(SEQ ID NO：22)	15nM
		SYBR I/carboxyrhodamine	1/100,000(0.1x)
Mg(OAc)2	3.0mM

Wherein "GLQDSE" refers to G46E L329A Q601R D640G S671F E678G CS5DNA polymerase.

The different reaction components included the following wild-type BRAF primers (labeled "F5W/R5W" in fig. 34):

F5W 5’-AATAGGTGATTTTGGTCTAGCTACAGU^＊-3’

R5W 5’-GGACCCACTCCATCGAGATTTCA^＊-3’

and the following mutant BRAF primers (labeled "F5M/R5M" in FIG. 34):

F5M 5’-AATAGGTGATTTTGGTCTAGCTACAGA^＊-3’

R5M 5’-GGACCCACTCCATCGAGATTTCU^＊-3’

wherein A is^＊Refers to 2 ' -phospho-A or 2 ' -monophosphate-3 ' -hydroxyadenosine nucleotide, and U^＊Is a 2 ' -phospho-U or 2 ' -monophosphate-3 ' -hydroxyuridine nucleotide (i.e., a 2 ' terminator nucleotide comprising a phosphate group at the 2 ' position).

In addition, each reaction was adjusted to 50. mu.l with DEPC treated water. The wild type reactant (labeled "WT" in fig. 34) contains the linearized DNA plasmid of the BRAF wild type sequence, and the mutant reactant (labeled "MT" in fig. 34) contains the linearized DNA plasmid of the BRAF mutant sequence. Negative reactions (labeled "NEG" in FIG. 34) contained HIV sample diluent (10mM Tris, 0.1mM EDTA, 20. mu.g/mL polyA, and 0.09% NaN₃) And does not contain DNA. The primer combination in the PCR yielded a 50bp amplicon. In addition, reactions were carried out using an ABI Prism 7700 sequence detector, using the following temperature conditions:

1 minute at 50 DEG C

1 minute at 93 DEG C

90 ℃ for 15 seconds

60 ℃ for 150 seconds → x60 cycles.

Example XIV: detection of fluorescent PAP release products

This prophetic example illustrates a real-time monitoring protocol involving PAP activation, in which a blocked primer results in the generation of a detectable signal as the primer is activated and extended.

Construction of 3' end, double-labeled oligonucleotide primers:

the primer QX described below is a DNA oligonucleotide comprising a quencher dye molecule attached to the 3' end of the 13 th nucleotide (A) of the Black Hole(BHQ)(BiosearchTechnologies，Inc.)。

The oligonucleotide primer for QX and the complementary oligonucleotide R1 (see below) were mixed in solution so that they formed a hybrid duplex. The duplex is further mixed with the reagents of Table IV provided below, including, inter alia, fluorescein-labeled deoxyriboadenine tetraphosphate (i.e., fluorescein-labeled 2' -terminator nucleotide) and a DNA polymerase capable of incorporating such labeled tetraphosphate. See U.S. patent application No.10/879,494, entitled "SYNTHESIS AND Compounds OF 2 '-TERMINATORNUCLEOTIDES", filed on 28.6.2004, and 10/879,493, filed on 28.6.2004, entitled "2' -TERMINATOR NUCLEOTIDE-RELATED METHODS AND ANDSTEMS". Incubation of the mixture at a temperature of 60 ℃ for e.g. 1 hour results in extension of the 3 ' end of the sequence QX by 1 nucleotide in a template-directed manner, resulting in extension of at least part of the QX oligonucleotides at their 3 ' end by fluorescein-labeled deoxyriboadenine 2 ' -phosphate nucleotides, denoted below as primer QX^FAM。

TABLE IV

Mixture component	Concentration of
		Zwitterionic buffer pH 8.3	50mM
KOAc	100mM
		Glycerol	8％(w/v)
Primer QX	10μM
		Oligonucleotide R1	15μM
Fluorescein dA4P	15μM
		G46E L329A E678G CS5DNA polymerase	50nM
Mg(OAc)2	2.5mM

Purification of the newly extended primer QX from the above mixture Using any number of purification methods known to those skilled in the art^FAM. Can purify the primer QX from the mixture^FAMAn example of such a method of (a) is High Performance Liquid Chromatography (HPLC). HPLC purification parameters were selected so that the primers QX^FAMThe preparation is substantially free of unextended primer QX and fluorescein-labeled adenine tetraphosphate. Double HPLC (reverse phase and anion exchange HPLC) is a known method for purifying such molecules.

Once purified, the molecule is, for example, a primer QX containing a BHQ quenching molecule and a fluorescein molecule on the same oligonucleotide^FAMA suppressed fluorescein signal is generally exhibited due to energy absorption by the BHQ2 "quencher" molecule.

Optionally, the primer QX is paired as described herein^FAMCarrying out chemical synthesis.

The sequences mentioned in this example are as follows:

primer QX 5 '-GCAAGCACCCTATCAQGGCAGTACCACA-3'

(wherein Q represents the presence of a BHQ molecule)

R1 3’-PCGTTCGTGGGATAGTCCGTCATGGTGTT-5’

(wherein P represents 3' phosphoric acid)

Primer QX^FAM 5’-GCAAGCACCCTATCA^QGGCAGTACCACA^F-3’

(wherein Q represents the presence of a BHQ molecule and F represents fluorescein-labeled 2' phosphoadenine)

Primer HC 25 '-GCAGAAAGCGTCTAGCCATGGCTTA-3'.

Application of primer in PCR

Primer QX as described above^FAMIn combination with the reagents in table V.

TABLE V

Components	Concentration of
		Zwitterionic buffer pH8.0	100mM
KOAc	100mM
		Glycerol	3.5％(v/v)
DMSO	5％(v/v)
		Primer QXFAM	150nM
Primer HC2	150nM
		dATP	200μM
dCTP	200μM
		dGTP	200μM
dTTP	30μM
		dUTP	300μM
UNG	1 unit of
		PPi	175μM
GLQDSE	15nM
		Target sequence	106Copy (a) of
Mg(OAc)2	3.0mM

In addition, each reaction was adjusted to 50. mu.l with DEPC treated water. Some reactions contain target sequences that serve as substrates for PCR amplification, while others do not. For example, the target sequence may be a DNA sequence identical to the 5' UTR region of the HCV genome. The combination of these primers in PCR is expected to produce an amplicon of approximately 244 bp.

The reaction was performed using an ABI Prism 7700 sequence Detector, using the following temperature conditions:

1 minute at 50 DEG C

1 minute at 93 DEG C

90 ℃ for 15 seconds

60 ℃ 150 "→ x60 cycles

For this PCR, a fluorescein-terminated primer QX is required^FAMAnd which will result in the removal of the fluorescein-labeled deoxyadenine tetraphosphate molecule. This release is expected to result in an increase in the fluorescence signal at a wavelength of about 520 nm. As the PCR progresses and the signal at a wavelength of about 520nm is monitored, one can expect to observe an increase in fluorescence in those reactions containing the target nucleic acid, while observing no fluorescence in those reactions not containing the targetAn increase in light.

Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all of the techniques and apparatus described above may be used in various combinations.

Claims (17)

1. An oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide of the formula:

wherein Z is O or CH₂；

B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof;

BG is a blocking group;

R₁h, OH, a hydrophilic group or a hydrophobic group;

x is a nucleotide or nucleotide analog;

n is an integer greater than 0; and

represents a single bond or a double bond.

2. The oligonucleotide or polynucleotide of claim 1, wherein the 2 ' -terminator nucleotide comprises a 2 ' -monophosphate-3 ' -hydroxyl nucleotide.

3. The oligonucleotide or polynucleotide of claim 1, wherein the 2' -terminator nucleotide is not extendable by one or more nucleotide incorporating biocatalysts selected from the group consisting of: G46E E678GCS 5DNA polymerase, G46E L329A E678G CS5DNA polymerase, G46E L329AD 640S671F CS5DNA polymerase, G46E L329A D640G S671F E678G CS5DNA polymerase, G46E E678E CS E DNA polymerase, Δ Z05E polymerase, E615E Taq DNA polymerase, yellow Thermus polymerase, TMA-25 polymerase, E678E TMA-25 polymerase, TMA-30 polymerase, E678E TMA-30 polymerase, Tth DNA polymerase, Thermus SPS-17 polymerase, E615E Taq polymerase, Thermus Z05E polymerase, T E DNA polymerase, Kornberg DNA I, Klinenosol DNA polymerase, Taq DNA polymerase, Micrococcus DNA polymerase, alpha DNA polymerase, reverse transcriptase, AMV RT-M RT polymerase, MuRT-RNA polymerase, DNA polymerase, t7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, incorporation of ribonucleotide DNA polymerase.

4. The oligonucleotide or polynucleotide of claim 1, wherein B comprises a formula selected from:

a)

wherein

X₁And X₂Independently selected from CR₈And N;

R₂is H, OH or NR₄R₅；

R₃Is H, OH or NR₆R₇；

R₄、R₅、R₆And R₇Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, aryloxy, and combinations thereof; and

R₈is H, halo, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, alkylol, alkenylalcohol, alkynylalcohol, unsubstituted polyethylene glycol or substituted polyethylene glycol;

b)

wherein

X₁And X₂Independently selected from CH and N;

R₂is O or S;

R₃is H, OH or NR₄R₅(ii) a And

R₄and R₅Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, and combinations thereof;

c)

wherein

R₂Is H, OH or NR₄R₅；

R₃Is H, OH or NR₆R₇(ii) a And

R₄、R₅、R₆and R₇Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, benzyl, aryl, and combinations thereof;

d)

wherein

X₁And X₂Independently selected from CH and N;

R₂selected from H, alkyl, alkenyl, benzyl, aryl, aryloxy, or combinations thereof; and

R₃is O or S;

e)

wherein

R₂And R₃Independently selected from O and S; and

R₄and R₅Independently selected from H, NH₂SH, OH, alkyl, alkenyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof;

f)

wherein

R₂And R₃Independently selected from O and S; and

R₄is H, NH₂SH, OH, alkyl, alkenyl, benzyl, aryl, aryloxy, alkoxy, halo, or combinations thereof;

g)

wherein R is₂And R₃Independently selected from O and S;

h)

wherein R is₂And R₃Independently selected from O and S, and

R₄is H, alkyl, alkenyl or alkynyl;

i)

wherein

R₂Is O or S;

R₃and R₄Independently selected from H, NH₂、SH、OH、COOH、COOCH₃、COOCH₂CH₃、CHO、NO₂CN, alkyl, alkenyl, alkynyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof; and

R₅is alkyl, alkenyl, aryl, benzyl, or combinations thereof;

j)

wherein

X is CH or N;

R₂and R₃Independently selected from H, OH and NHR₄；

R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; and

R₅is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, or combinations thereof;

k)

wherein

X is CH or N;

R₂is O or S;

R₃is H, OH or NHR₄；

R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; and

R₅is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, or combinations thereof.

5. The oligonucleotide or polynucleotide of claim 1, wherein BG is selected from: CN, NO₂、N₃Halogen group, ether group, aldehyde group, carboxylic acid group, ester group, amino group, OCH₃、OCH₂COOH, O-silyl ether groups, ketone groups, O-lactone groups, O-alkyl groups, O-cycloalkyl groups, O-alkenyl groups, O-alkynyl groups, carbamate groups, imide groups, amide groups, and combinations thereof.

6. The oligonucleotide or polynucleotide of claim 1, wherein BG comprises the formula:

or

Wherein

Q is O, S or NH;

x is H, OH, CH₃、BH₃F or SeH; and

z is O, S or Se; or

Wherein

Q is O, S or NH;

x is O, S or NH;

z is O, S or Se; and

r is alkyl, alkenyl or alkynyl; or

Wherein

Q is O, S or NH;

x is O, S or NH;

z is O, S or Se;

l is-CONH (CH)₂)_nNH-、-CO(CH₂)_nNH-or-CONH (CH)₂CH₂O)_nCH₂CH₂NH-；

n is an integer greater than 0; and

r is NH₂SH, COOH, quencher moieties, reporter moieties, biotin or affinity moieties.

7. A method of preparing an oligonucleotide or polynucleotide, the method comprising:

(a) providing a nucleic acid synthesis reagent comprising a 2' -terminator nucleotide of the formula:

wherein

Z is O or CH₂；

B is at least one homocyclic ring, at least one heterocyclic ring, at least one aryl group, or a combination thereof;

BG is a blocking group;

r is O, NH or S;

PG is a protecting group;

x is a nucleotide or nucleotide analog;

n is an integer greater than 0;

represents a single or double bond; and

(b) removing PG from the nucleic acid synthesis reagent to produce an oligonucleotide comprising a 2' -terminator nucleotide of the formula:

wherein R is₁H, OH, a hydrophilic group or a hydrophobic group, thereby preparing the oligonucleotide or polynucleotide.

8. The method of claim 7, wherein the 2 ' -terminator nucleotide comprises a 2 ' -monophosphate-3 ' -hydroxyl nucleotide.

9. The method of claim 7, wherein the 2' -terminator nucleotide is not extendable by one or more nucleotide incorporating biocatalysts selected from the group consisting of: G46E E678G CS5DNA polymerase, G46E L329A E678G CS5DNA polymerase, G46E L329A D640G S671F CS5DNA polymerase, G46E L329A D640G S671F E678G CS5DNA polymerase, G46EE678G CS6DNA polymerase, Δ Z05 6 polymerase, E615 6 Taq DNA polymerase, Thermus flavus polymerase, TMA-25 polymerase, E678 6 TMA-25 polymerase, TMA-30 polymerase, E678 6 TMA-30 polymerase, Tth DNA polymerase, Thermus SPS-17 polymerase, E615GTaq polymerase, Thermus Z05 6 polymerase, T7DNA polymerase, Kornberg DNA I, Klineol DNA polymerase, Taq DNA polymerase, Micrococcus DNA polymerase, alpha DNA polymerase, reverse transcriptase, AMV RT-M polymerase, MuRT-RNA polymerase, DNA polymerase, t7 RNA polymerase, RNA polymerase II, terminal transferase, polynucleotide phosphorylase, incorporation of ribonucleotide DNA polymerase.

10. The method of claim 7, which comprises attaching nucleic acid synthesis reagents or components thereof to a solid support prior to step (b).

11. An oligonucleotide or polynucleotide prepared by the method of claim 7.

12. The process of claim 7, wherein the protecting group is selected from the group consisting of: trityl, monomethoxytrityl, dimethoxytrityl, jerusalem artichoke glycosyl, fluorenylmethoxycarbonyl and benzhydryloxycarbonyl.

13. The method of claim 7, wherein the protecting group comprises the formula:

or

14. The method of claim 7, wherein B comprises a formula selected from:

a)

wherein

X₁And X₂Independently selected from CR₈And N;

R₂is H, OH or NR₄R₅；

R₃Is H, OH or NR₆R₇；

R₄、R₅、R₆And R₇Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, aryloxy, and combinations thereof; and

R₈is H, halo, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, alkylol, alkenylalcohol, alkynylalcohol, unsubstituted polyethylene glycol or substituted polyethylene glycol;

b)

wherein

X₁And X₂Independently selected from CH and N;

R₂is O or S;

R₃is H, OH or NR₄R₅(ii) a And

R₄and R₅Independently selected from the group consisting of H, alkyl, alkenyl, benzyl, aryl, and combinations thereof;

c)

wherein

R₂Is H, OH or NR₄R₅；

R₃Is H, OH or NR₆R₇(ii) a And

R₄、R₅、R₆and R₇Independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, benzyl, aryl, and combinations thereof;

d)

wherein

X₁And X₂Independently selected from CH and N;

R₂selected from H, alkyl, alkenyl, benzyl, aryl, aryloxy, or combinations thereof; and

R₃is O or S;

e)

wherein

R₂And R₃Independently selected from O and S; and

R₄and R₅Independently selected from H, NH₂SH, OH, alkyl, alkenyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof;

f)

wherein

R₂And R₃Independently selected from O and S; and

R₄is H, NH₂SH, OH, alkyl, alkenyl, benzyl, aryl, aryloxy, alkoxy, halo, or combinations thereof;

g)

wherein R is₂And R₃Independently selected from O and S;

h)

wherein R is₂And R₃Independently selected from O and S, and

R₄is H, alkyl, alkenyl or alkynyl;

i)

wherein

R₂Is O or S;

R₃and R₄Independently selected from H, NH₂、SH、OH、COOH、COOCH₃、COOCH₂CH₃、CHO、NO₂CN, alkyl, alkenyl, alkynyl, benzyl, aryl, aryloxy, alkoxy, halo, and combinations thereof; and

R₅is alkyl, alkenyl, aryl, benzyl, or combinations thereof;

j)

wherein

X is CH or N;

R₂and R₃Independently selected from H, OH and NHR₄；

R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; and R₅Is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, or combinations thereof;

k)

wherein

X is CH or N;

R₂is O or S;

R₃is H, OH or NHR₄；

R₄Is H, alkyl, alkenyl, benzyl, aryl, or combinations thereof; and

R₅is OH, NH₂SH, halo, ether, thioether, alkyl, alkenyl, alkynyl, alkylamino, alkenylamino, alkynylamino, or combinations thereof.

15. The method of claim 7, wherein BG is selected from: CN, NO₂、N₃Halogen group, ether group, aldehyde group, carboxylic acid group, ester group, amino group, OCH₃、OCH₂COOH, O-silyl ether groups, ketone groups, O-lactone groups, O-alkyl groups, O-cycloalkyl groups, O-alkenyl groups, O-alkynyl groups, carbamate groups, imide groups, amide groups, and combinations thereof.

16. The method of claim 7, wherein BG comprises the formula:

or

Wherein

Q is O, S or NH;

x is H, OH, CH₃、BH₃F or SeH; and

z is O, S or Se; or

Wherein

Q is O, S or NH;

x is O, S or NH;

z is O, S or Se; and

r is alkyl, alkenyl or alkynyl; or

Wherein

Q is O, S or NH;

x is O, S or NH;

z is O, S or Se;

l is-CONH (CH)₂)_nNH-、-CO(CH₂)_nNH-or-CONH (CH)₂CH₂O)_nCH₂CH₂NH-；

n is an integer greater than 0; and

r is NH₂SH, COOH, quencher moieties, reporter moieties, biotin or affinity moieties.

17. A kit comprising one or more of:

(a) instructions for preparing an oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide; or

(b) At least one oligonucleotide or polynucleotide comprising a 2' -terminator nucleotide.