Reagents and methods for post-synthetic modification of nucleic acids

Abstract

The present invention relates to compositions and methods (reagents and protocols) for post-synthetic modification of nucleic acids using labels (e.g., fluorescent dyes) obtained from solid phase oligonucleotide synthesis. The coupling agent is a triazine-based salt 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium (DMT-MM) in the presence of a counter anion.

Description

Technical Field

[0001] The present invention relates generally to the field of nucleic acid chemistry and, more particularly, to methods and compositions for post-synthetic modification of nucleic acids with labels using coupling agents. Background Art

[0002] Many procedures used in biomedical research and recombinant DNA technology rely on the use of labeled nucleotides or polynucleotide derivatives. In order for modified nucleotides to be suitable as labeled forms of naturally occurring nucleotides, several criteria must generally be met. First, the modified compound must contain a unique substituent or probe (i.e., not usually associated with a nucleotide or polynucleotide). Second, the probe must react specifically with a chemical or biological reagent to provide a sensitive detection system. Third, the analog must be an effective substrate for the nuclease commonly studied, because many practical applications require that the analog be enzymatically metabolized (e.g., the analog must serve as a substrate for a nucleic acid polymerase). To this end, the probe moiety should not be placed in a ring position that spatially or otherwise interferes with the normal Watson-Crick hydrogen bonding potential of the base. In this case, the substituent may produce a compound that is inactive as a polymerase substrate. Fourth, the detection system should be able to interact with probe substituents incorporated into single-stranded and double-stranded polynucleotides to be compatible with nucleic acid hybridization methods. Fifth, the physical and biochemical properties of polynucleotides containing a small amount of probe substituents should not be significantly changed, thereby eliminating the need for extensive modifications to current procedures using hybridization probes. This criterion must be met whether the probe is introduced enzymatically or by direct chemical methods.Finally, the ligation of the attached probe moiety should withstand all experimental conditions to which normal nucleotides and polynucleotides are routinely subjected (e.g., extended hybridization at elevated temperatures, phenol and organic solvent extraction or electrophoresis).

[0003] In modern life sciences, methods for labeling biomolecules with fluorescent, luminescent, quenching or phosphorescent dyes or affinity tags have become indispensable methods, and it is important for these conjugation strategies that they allow site-specific postsynthetic coupling of complex molecules under mild conditions. Although N-hydroxysuccinimide (NHS) ester chemistry has dominated the labeling of protein and nucleic acid molecules, simplicity and cost considerations have prompted the need for new labeling methods that should be applicable to small chemically synthesized oligonucleotides as well as longer enzymatically amplified DNA chains.

[0004] In recent years, triazine-based compounds (Kunishima et al., Tetrahedron 55, 13159-13170, 1999), which were originally developed for natural product and peptide synthesis, have attracted attention as effective coupling agents and found their application in amidation, esterification, glycosidation and phosphonylation reactions. Although 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMT-MM) is highly reactive towards the condensation of carboxyl and amino groups to form amides, it is extremely stable in water and ethanol. Therefore, DMT-MM is an attractive alternative to other water-compatible reagents such as the carbodiimide EDC [N-(3-dimethyl-aminopropyl)-N′-ethylcarbodiimide], which can react with phosphate and easily decomposes at low pH, or the peptide coupling agent TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate). Summary of the invention

[0005] The present invention provides a method for post-synthetic modification of nucleic acids obtained by solid phase oligonucleotide synthesis using 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium (DMT-MM) salt as a coupling agent and a label (such as a fluorescent dye). The chemical structure of DMT-MM is as follows Figure 1 As shown. Therefore, on the one hand, the present invention relates to a method for post-synthetic modification of nucleic acids using a label, the method comprising the following steps: (a) preparing an amino-modified nucleic acid molecule comprising one or more amino modifications; (b) activating a carboxyl-modified label using DMT-MM; and (c) reacting the amino-modified nucleic acid molecule with the activated carboxyl-modified label to produce a labeled nucleic acid molecule. In one embodiment, the activation step involves DMT-MM in the presence of a counter-anion. In one embodiment, the counter-anion is a weakly coordinating or non-coordinating anion, wherein the weakly coordinating or non-coordinating anion is not a carboxylic acid. In one embodiment, the counter-anion or the weakly coordinating or non-coordinating anion is selected from the group consisting of: tetrafluoroborate (BF4 - ), hexafluorophosphate (PF6 - ), benzenesulfonate (CH5SO3 - ), bis(trifluoromethanesulfonyl)imide((N[SO2(CF3)]2) - ), bromide ion (Br - ), 10-camphorsulfonate, chloride ion (Cl - ), iodide ion (I - ), methanesulfonate (methanesulfonate, CH3SO3 - ), perchlorate (ClO4 - ), phosphate (PO4 3- ), sulfate (SO42- ), tetrachloroaluminate (AlCl4 - ), tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (B[3,5-(CF3)2C6H3]4 - ), tetrakis(hexafluoroisopropyl)aluminateAl[OC(CF3)3]4 - ), tetrakis(pentafluorophenyl)borate [B(C6F5)4 - ], p-toluenesulfonate (toluenesulfonate, CH3C6H4SO3 - ), trifluoromethanesulfonate (trifluoromethanesulfonate, CF3SO3 - In one embodiment, the counter anion or weakly coordinating or non-coordinating anion is tetrafluoroborate (BF4 - ) or hexafluorophosphate (PF6 - ). In one embodiment, the nucleic acid molecule is an oligonucleotide. In another embodiment, the oligonucleotide is between 3 and 500 nucleotides in length. In another embodiment, the 5′ or 3′ end of the oligonucleotide is amino-modified. In another embodiment, the 5′ and 3′ ends of the oligonucleotide are amino-modified. In another embodiment, the oligonucleotide is internally amino-modified. In yet another embodiment, the label is selected from the group consisting of a fluorescent dye, a luminescent dye, a quenching dye, a phosphorescent dye, and an affinity tag. In one embodiment, the label is a fluorescent dye. In another embodiment, the fluorescent dye is essentially incompatible with phosphoramidite-based DNA synthesis. In another embodiment, the fluorescent dye comprises a sulfonate moiety. In one embodiment, if the length of the oligonucleotide is less than 100 nucleotides, the stoichiometry of the amino-modified oligonucleotide and the activated carboxyl-modified label is 1:3 or between 1:3 and 1:1.5. In another embodiment, if the length of the oligonucleotide is greater than 100 nucleotides, the activated carboxyl-modified label is used in a 2-20-fold molar excess of the amino-modified oligonucleotide. In one embodiment, the oligonucleotide is synthesized using phosphoramidite-based DNA synthesis. In one embodiment, the oligonucleotide is purified after synthesis. In another embodiment, the oligonucleotide is not purified after synthesis. In one embodiment, the method of steps is performed during the synthesis of the oligonucleotide, wherein the oligonucleotide is bound to a solid support. In one embodiment, the nucleic acid molecule is single-stranded. In another embodiment, the nucleic acid molecule is double-stranded. In another embodiment, the nucleic acid molecule is the product of enzymatic amplification. In another embodiment, the nucleic acid molecule is selected from the group consisting of DNA, RNA, LNA, PNA, nucleic acid analogs, and mixtures of DNA, RNA, LNA, PNA, and nucleic acid analogs.

[0006] The present invention also provides a labeled nucleic acid, which is labeled by any of the above methods, wherein the method comprises the following steps: (a) preparing an amino-modified nucleic acid molecule, which includes one or more amino modifications; (b) using DMT-MM to activate a carboxyl-modified marker; and (c) reacting the amino-modified nucleic acid molecule with an activated carboxyl-modified marker to produce a labeled nucleic acid molecule. In some embodiments, the labeled nucleic acid is an oligonucleotide. In some embodiments, the length of the oligonucleotide is less than 100 nucleotides. In some embodiments, the labeled oligonucleotide is a primer oligonucleotide. In other embodiments, the labeled oligonucleotide is a probe oligonucleotide.

[0007] The present invention also provides a kit, which includes a nucleic acid labeled by any of the above methods, wherein the method comprises the following steps: (a) preparing an amino-modified nucleic acid molecule, which includes one or more amino modifications; (b) using DMT-MM to activate a carboxyl-modified marker; and (c) reacting the amino-modified nucleic acid molecule with an activated carboxyl-modified marker to produce a labeled nucleic acid molecule. In some embodiments, the labeled nucleic acid is an oligonucleotide. In some embodiments, the length of the oligonucleotide is less than 100 nucleotides. In some embodiments, the labeled oligonucleotide is a primer oligonucleotide. In other embodiments, the labeled oligonucleotide is a probe oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Shown is the structure of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMT-MM) and an example of its choice of counter anion.

[0009] Figure 2A UPLC analysis of the 5' labeling reaction is shown. The 5' end of the unpurified oligonucleotide obtained by solid phase DNA synthesis was labeled with Dy380XL or Dy395XL carboxylic acid. Both the 30mer (162071) and 33mer (162073, 162074) oligonucleotides included a 3'-BHQ-2 quencher. The reaction conversion (%) was determined by comparing the peak areas of the starting material (SM) and the product (P).

[0010] Figure 2B Overlapping absorption spectra of oligonucleotides before and after labeling with Dy380XL (upper panel) and Dy395XL (lower panel) are shown. Spectra extracted from Figure 2A The upper two chromatograms (162071A and B).

[0011] Figure 3 UPLC analysis of a 5′ labeling reaction using Dy395XL, a fluorescent dye with very low solubility in organic solvents, is shown. Figure 2AThe same as sample number 162074B in , but using 50% water in the pre-activation step. The reaction conversion increased from 69.8% to 80.4%, which was determined by comparing the peak areas of the starting material (SM) and the product (P).

[0012] Figure 4A UPLC chromatograms of the 5′ labeling reaction at two time points (1.0 h, 3.0 h) are shown. The 5′ end of the unpurified 29mer oligonucleotide was purified by Atto490LS and Chromeo TM 494 carboxylic acid labeling. Both oligonucleotides contained a 3′-BHQ-2 quencher. The reaction conversion (%) was determined by comparing the peak areas of the starting material (SM) and the product (P).

[0013] Figure 4B Shown in use Atto490LS (above) and Chromeo TM 494 (bottom) Overlapping absorption spectra of oligonucleotides before and after labeling. Spectra extracted from Figure 4A The first and third chromatograms in (162072B and A).

[0014] Figure 5 A representative example of labeling a DNA oligonucleotide sequence at an internal position is shown. Shown is a UPLC chromatogram of a pre-purified 55mer DNA-LNA hybrid oligonucleotide (161130) using a 5′-BHQ-2 quencher, which was labeled with C343 carboxylic acid. Samples were taken before the reaction (reference) and 1.0 h and 3.0 h after the reaction. The reaction conversion (%) was determined by comparing the peak areas of the starting material (SM) and the product (P).

[0015] Figure 6 The final UPLC analysis of DNA oligonucleotide probes obtained by labeling reaction with fluorescent dye using DMT-MM and subsequent purification is shown. The lengths of the DNA oligonucleotide sequences are 30mer (162071), 29mer (162072) and 33mer (162073, 162074), respectively, each of which contains a 3′-BHQ-2 quencher.

[0016] Figure 7 Growth curves generated from the quantitative PCR (qPCR) experiments described in Example 2 are shown.

[0017] Figure 8 The threshold cycle (C t ) value, fluorescence baseline, signal amplitude and experimental rate constant value (K exp ) table.

[0018] Fig.9A A representative example of a large-scale labeling reaction using DMT-MM as described in Example 3 is shown. A 33mer oligonucleotide (171742) with a 3′-BHQ-2 quencher was obtained from solid phase DNA synthesis at a scale of 15 μmol and labeled with Dy396XL at the 5′ end without prior purification. The first two panels show UPLC chromatograms of the DNA before and after 15 min of reaction. Comparison of the peak areas of the raw material and the product indicates that ˜95% is converted to the product. The third panel shows the overlapping absorption spectra of the raw material and the product.

[0019] Fig. 9B Shows the reproducibility of large-scale DNA labeling reactions. Fig.9A UPLC chromatograms of reactions with the same DNA sequence and under the same conditions, but with different fluorescent dye synthesis batches.

[0020] Fig. 10A Shown are UPLC chromatograms of purified DNA oligonucleotide probes obtained from six independent large-scale labeling reactions performed using DMT-MM as described in Example 3. The oligonucleotide sequences were 30mer (171742, 171743, 171744) and 33mer (171744, 171745, 171747) in length, each of which contained a 3′-BHQ-2 quencher.

[0021] Fig. 10B Mass spectrometry analysis of purified DNA oligonucleotide probes obtained by large-scale labeling reaction using DMT-MM described in Example 3 is shown.

[0022] Fig.11 The threshold cycle (C t ) value, fluorescence baseline, signal amplitude and experimental rate constant value (K exp For each qPCR, two Dy396XL-labeled probe sequences were combined and hybridized to two different regions of the target DNA sequence. DETAILED DESCRIPTION

[0023] definition

[0024] Unless otherwise indicated, all scientific and technical terms used herein have the same meaning as those of ordinary skill in the art to which the present invention belongs. Although substantially any method and material similar to the method and material described herein can be used in the practice or testing of the present invention, only exemplary methods and materials are described. For purposes of the present invention, the following terms are defined as follows.

[0025] The terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0026] As used herein and unless otherwise indicated, the term "alkyl" refers to a saturated, monovalent, straight or branched hydrocarbon chain. Examples of alkyl include, but are not limited to, C1-C6 alkyl groups, such as methyl, ethyl, propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl and hexyl, and longer alkyl groups, such as heptyl and octyl. An alkyl group can be unsubstituted or substituted with one or two suitable substituents.

[0027] As used herein and unless otherwise indicated, the term -O-alkyl (or alkyl-O-) refers to an "alkoxy" group, wherein alkyl is as defined above. The alkoxy group may be unsubstituted or substituted with one or more suitable substituents. The length of the alkyl chain of the alkoxy group may be, for example, 1 to 6 carbon atoms.

[0028] As used herein and unless otherwise indicated, the term "metal" refers to a Group I or Group II metal, including but not limited to Li + 、Na + Mg 2+ or Mn 2+ .

[0029] As used herein and unless otherwise indicated, the terms "linker" and "joint" are used interchangeably and refer to the portion of a detectable label that is capable of covalently binding a base to a label, e.g., forming a "link" that connects a nucleoside, nucleotide, or nucleic acid to a label. Examples of joints include, but are not limited to, O, S, or NH. Optionally, the linker or joint is a covalent bond (i.e., the label is covalently bound to the base).

[0030] As used herein and unless otherwise indicated, the term "counterion" or "counteranion" refers to an ion that is stable and synthetically available. Examples of counteranions are Figure 1 As shown, and include but are not limited to tetrafluoroborate (BF4 - ), hexafluorophosphate (PF6 - ), benzenesulfonate (CH5SO3 - ), bis(trifluoromethanesulfonyl)imide((N[SO2(CF3)]2) -), bromide ion (Br - ), 10-camphorsulfonate, chloride ion (Cl - ), iodide ion (I - ), methanesulfonate (methanesulfonate, CH3SO3 - ), perchlorate (ClO4 - ), phosphate (PO4 3- ), sulfate (SO4 2- ), tetrachloroaluminate (AlCl4 - ), tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (B[3,5-(CF3)2C6H3]4 - ), tetrakis(hexafluoroisopropyl)aluminate (Al[OC(CF3)3]4 - ), tetrakis(pentafluorophenyl)borate [B(C6F5)4 - ], p-toluenesulfonate (toluenesulfonate, CH3C6H4SO3 - ) and trifluoromethanesulfonate (trifluoromethanesulfonate, CF3SO3 - ). The counter anion can be any weakly coordinating or non-coordinating anion.

[0031] As used herein, the term "non-coordinating anion" is used interchangeably with the term "weakly coordinating anion" and refers to an anion that is not coordinated to the cation or is only weakly coordinated to the cation, thereby maintaining sufficient instability to be replaced by a neutral Lewis base. "Compatible" non-coordinating anions are those ions that are not degraded to neutrality when the initially formed complex decomposes. In addition, the anion does not transfer anionic substituents or fragments to the cation, thereby forming a neutral transition metal compound of the anion and neutral byproducts from the anion. Non-coordinating anions useful according to the present invention are compatible, stabilizing the DMT-MM cation in the sense of balancing its ionic charge to +1, but still retaining sufficient instability to allow displacement during the reaction.

[0032] As used herein, a "biocompatible" material is non-toxic as used and has no substantial deleterious effects on biological molecules.

[0033] As used herein and unless otherwise indicated, the term "nucleobase" refers to adenine, cytosine, guanine, thymine, or uracil.

[0034] As used herein and unless otherwise indicated, the term "nucleobase analogue" refers to a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring capable of forming Watson-Crick hydrogen bonds with a complementary nucleobase or nucleobase analogue. Preferably, the nucleobase analogue is a purine, a deazapurine or a pyrimidine. Exemplary nucleobase analogues include, but are not limited to, 7-deazaadenine, inosine, nebularine, nitropyrrole, nitroindole, 2-aminopurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, 5-propynylcytidine, isocytidine, isoguanine, 7-deazaguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, 0 6 -methylguanine, N 6 -methyladenine, O 4 -methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole, ethynyl adenine, etc. Other exemplary nucleobase analogs can be found in Fasman, 1989, Practical Handbook of Biochemistry and Molecular Biology, pages 385-394, CRC Press, Boca Raton, FL, and references cited therein.

[0035] As used herein and unless otherwise indicated, the term "nucleoside" refers to a compound consisting of a nucleobase covalently linked to the C1' carbon of a substituted or unsubstituted ribose sugar. Typical substituted ribose sugars include, but are not limited to, those in which one or more carbon atoms, preferably one, most preferably the 3' carbon atom, are substituted with one or more of the same or different -R, -OR, -NRR or halogen groups, wherein each R is independently -H, (C1-C6)alkyl or (C5-C 14 ) aromatic group. Particularly preferred ribose sugars are ribose, 2′-deoxyribose, 2′,3′-deoxyribose, 3′-haloriboses, 3′-fluororibose, 3′-chlororibose, 3′-alkylribose, and the like. When the nucleobase is A or G, the ribose is attached to the N 9 When the nucleobase is C, T, or U, the pentose is attached to the N 1 Position (see, e.g., Kornberg and Baker, 1992, DNA Replication, 2nd ed., Freeman and Company, San Francisco).

[0036] As used herein and unless otherwise indicated, the term "nucleoside analogs" refers to nucleosides in which a nucleobase, a ribose or both are replaced by their respective analogs. Exemplary nucleobase analogs are those previously defined. Exemplary ribose sugar analogs include, but are not limited to, substituted or unsubstituted furanoses, for example, erythrose and hexoses and substituted or unsubstituted 3-6 carbon acyclic sugars, each ring having more than or less than five members. Typical substituted furanosidases and acyclic sugars are those in which one or more carbon atoms are replaced by one or more identical or different-R, -OR, NRR or halogen groups, wherein R is each independently H, (C1-C6) alkyl or (C5-C6) alkyl. 14 )aryl.

[0037] As used herein and unless otherwise indicated, the term "nucleotide" refers to a nucleoside in which one or more (usually one) ribose carbons are substituted with a phosphate having the formula:

[0038]

[0039] wherein a is an integer between 0 and 4. Preferably, a is 2 and the phosphate is attached to the 3′ or 5′-carbon of the ribose, e.g., ribose 3′-triphosphate, 2′-deoxyribose 3′-triphosphate, ribose 5′-triphosphate, 2′-deoxyribose 5′-triphosphate, 3′-haloriboses 5′-triphosphate, 3′-alkylriboses 5′-triphosphate, 2′,3′-dideoxyribose 5′-triphosphate, etc.

[0040] As used herein and unless otherwise indicated, the terms "nucleotide derivatives" and "nucleotide analogs" can be used interchangeably and refer to nucleotides in which nucleotides, ribose and / or one or more phosphates are replaced by their respective analogs. Exemplary nucleobases and ribose analogs are those previously described as conjugated to nucleoside analogs. Exemplary phosphate analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phospholinoleate, phosphodiselenoate, phosphatoanilide, phosphatoaniline, phosphatocarbamate, boronate phosphorothioate, peptide nucleic acid (PNA) monomers, etc., including any associated counter-agents (if present).

[0041] As used herein and unless otherwise indicated, the term "nucleoside or nucleotide" refers to nucleosides and / or nucleotides and / or mixtures thereof.

[0042] As used herein and unless otherwise indicated, the term "nucleic acid" refers to a linear polymer chain of nucleoside monomer units, which are covalently linked to each other by phosphate internucleotide bonds. Unless otherwise indicated, "nucleic acid" used herein includes polymers of any length, including oligonucleotides, nucleic acids, and nucleic acids of terms commonly used in the art. Therefore, the size range of nucleic acids according to the present invention can be from several monomer units (for example, 4 to 40), to hundreds of monomer units, to thousands of monomer units, or even more monomer units. Such nucleic acids can also be described herein with their functions, such as primers or probes. Whenever nucleic acid is represented by a letter sequence such as "ATGCCTG", it should be understood that the sequence is presented in a 5'→3' direction. Unless otherwise indicated, the nucleic acid of the sequence described herein is a 2'-deoxyribonucleic acid.

[0043] "Oligonucleotides" and "modified oligonucleotides" according to the present invention can be synthesized as mainly described in the art and known to those skilled in the art. Methods for preparing oligomeric compounds with specific sequences are known in the art and include, for example, cloning and limiting appropriate sequences and direct chemical synthesis. Chemical synthesis methods can include, for example, the phosphotriester method described in Narang, SA, et al., Methods in Enzymology 68 (1979) 90-98, the phosphodiester method disclosed in Brown, EL, et al., Methods in Enzymology 68 (1979) 109-151, the phosphoramidite method disclosed in Beaucage et al., Tetrahedron Letters 22 (1981) 1859 and U.S. Patent No. 4,415,732, Garegg, et al., H-phosphonate method disclosed in Chem. Scr. 25 (1985) 280-282, and the solid support method disclosed in U.S. Patent No. 4,458,066.

[0044] As used herein and unless otherwise indicated, the term "nucleic acid analog" refers to a nucleic acid in which at least one nucleoside monomer unit is a "nucleoside analog" and / or at least one phosphate internucleotide bond is a phosphate analog, as defined above in "nucleotide analogs". Preferred classes of nucleic acid analogs are those in which the sugar and internucleotide bonds are replaced by uncharged neutral amide groups, such as carbamate morpholinos and peptide nucleic acids ("PNAs"). Preferred PNAs are those with an N-(2-aminoethyl)glycine amide backbone (see, e.g., Nielsen et al., 1991, Science 254: 1497-1500).

[0045] As used herein, the term "amino-modified oligonucleotide" refers to an oligonucleotide modified with one or more amino groups that can react with a carboxyl moiety (e.g., a carboxyl moiety on a label). In contrast, the term "carboxyl-modified oligonucleotide" refers to an oligonucleotide modified with one or more carboxyl groups that can react with an amino moiety (e.g., an amino moiety on a label). Therefore, according to the purposes of the present invention, for the purpose of labeling, the amino and carboxyl functional groups can be reversed.

[0046] The amino modification may include the addition of various moieties including one or more primary or secondary amines, or the modification may include the addition of one or more primary or secondary amines. Thus, the amino modification may include the general formula [O]-Z n , wherein [O] is an oligonucleotide; Z is selected from NH2, NHR, RNH2 or RNHR at each occurrence; n is a non-zero integer; and R is independently and each time selected from a hydrocarbyl or substituted hydrocarbyl group. For the sake of clarity, it should be understood that when R is a divalent group, the divalent R group can be a hydrocarbylene or substituted hydrocarbylene group, including but not limited to an alkylene or substituted alkylene group. When R is a monovalent group, R can be any number of hydrocarbyl or substituted hydrocarbyl groups, including but not limited to alkyl and substituted alkyl groups. When it is a monovalent group, R can also be any number of protecting groups generally known in the art, such as those described in Protecting Groups in Organic Synthesis (Green, Wuts; 3rd Edition, 1999, John Wiley & Sons, Inc.). The oligonucleotide can be amino-modified at any position within the oligonucleotide. In some embodiments, the amino modification is at the 5' end or the 3' end. In other embodiments, the amino modification is at one or more internal positions within the oligonucleotide. Oligonucleotides including amino-modified ends can be prepared using methods and reagents commonly used in the art. For example, oligonucleotides having an amino modification at the 5′ end can be prepared using various linkers (also referred to as modifiers in the art), including but not limited to C2-C 12 An amino linker or an amino-protected derivative thereof. 12Examples of amino linkers or amino-protected derivatives thereof include, but are not limited to, 2-[2-(4-monomethoxytrityl)aminoethoxy]ethyl(2-cyanoethyl)-N,N-diisopropyl-)phosphoramidite (C5 MMT amino linker), 6-(4-monomethoxytritylamino)hexyl[(2-cyanohexyl)-(N,N-diisopropyl)]phosphoramidite (C6MMT amino linker), 6-(trifluoroacetylamino)hexyl[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (C6TFA amino linker), 7-(4-monomethoxytritylamino)heptyl[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (C7MMT amino linker), 12-(4-monomethoxytritylamino)dodecyl[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (C8MMT amino linker), 12 MMT amino linker). In other embodiments, the oligonucleotide includes one or more amino modifications at a position other than the 3′ or 5′ terminus. For example, the oligonucleotide may include an amino modification group directly attached or otherwise tethered to one or more bases of the oligonucleotide. Such amino-modified oligonucleotides may be prepared using any number of phosphoramidites generally known in the art, including but not limited to: amino C6 dT (e.g., 5′-dimethoxytrityl-5-[N-(trifluoroacetamidohexyl)-3-acryloyl imino]-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)] phosphoramidite, 5′-dimethoxytrityl-5-[N-((9-fluorenyl-methoxycarbonyl)-aminohexyl)-3-acryloyl-imino]-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)] phosphoramidite, etc.); amino C6 dA (e.g., 5′-dimethoxytrityl-N 6 -Benzoyl-N 8 -[6-(trifluoroacetylamino)-hexan-1-yl]-8-amino-2′-deoxyadenosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite); aminoC6 dG (e.g., 5′-dimethoxytrityl-N2-(N,N-dimethylaminomethylene)-N 8-[6-(trifluoroacetylamino)-hexan-1-yl]-8-amino-2′-deoxyguanosine-3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite), aminoC6dC (e.g., 5′-dimethoxytrityl-N-dimethylformamidine-5-[N-(trifluoroacetylaminohexyl)-3-acrylimide]-2′-deoxycytidine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite), and / or aminoC2dT (e.g., 5′-dimethoxytrityl-5-[N-(trifluoroacetylaminoethyl)-3-acrylimide]-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite). For clarity, it should be understood that following synthesis of the amino-modified oligonucleotide, any phosphoramidite comprising a protected amino group, including but not limited to those described above, may be optionally deprotected using deprotection methods known in the art.

[0047] As used herein, unless otherwise indicated, the term "protecting group" refers to a group that is reversibly attached to a hydroxyl or amine moiety that renders the hydroxyl or amine moiety unreactive in subsequent reactions and that can be selectively cleaved to regenerate the hydroxyl or amine moiety once the protection purpose is achieved. Examples of protecting groups can be found in Greene, TW, Protective Groups in Organic Synthesis, 3rd edition (1999). In one embodiment, the protecting group has stability in alkaline reaction medium, but can be cleaved by acid. Examples of base-stable, acid-labile protecting groups suitable for use in the present invention include, but are not limited to, ethers such as methyl, methoxymethyl, methylthiomethyl, methoxyethoxymethyl, bis(2-chloroethoxy)methyl, tetrahydropyranyl, tetrahydrothiopyranyl, tetrahydrofuranyl, tetrahydrothiofuranyl, 1-ethoxyethyl, 1-methyl-1-methoxyethyl, tert-butyl, allyl, benzyl, o-nitrobenzyl, trityl, α-naphthyldibenzhydryl, p-methoxyphenyl-dibenzhydryl, 9-(9-phenyl-10-oxo)anthryl, trimethylsilyl, isopropyldimethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, tribenzylsilyl, and triisopropylsilyl; and esters such as pivalate, adamantanoate, and 2,4,6-trimethylbenzoate.

[0048] As used herein and unless otherwise indicated, the term "label" refers to a detectable molecule or atom that is covalently or non-covalently attached to a nucleoside or nucleotide, a nucleoside or nucleotide analog, a nucleic acid, a nucleic acid analog, or a terminator. In one embodiment, a nucleoside or nucleotide, a nucleoside or nucleotide analog, a nucleic acid, a nucleic acid analog, or a terminator has a detectable label covalently attached to a core base. The term "label" can also refer to a molecule that regulates the detection of another detectable label (such as a quencher). As used herein, the term "detectable label" is intended to include not only "directly" detected molecules or labels (e.g., chromophores or fluorophores), but also "indirectly" detected parts (e.g., biotin), which are combined with a second, third, or higher binding partner (e.g., avidin or streptavidin), one of which carries a "direct" label.

[0049] Other examples of labels include fluorescent compounds that become detectable due to fluorescence when exposed to light of an appropriate wavelength and are detected and / or measured by microscopy or fluorescence. Commonly used fluorescent labeling compounds include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, gamma-benzaldehyde, and fluorescamine. Detectable labels can be metals that emit fluorescence, such as 152 Eu, or other metals of the lanthanide series can be attached to the oligonucleotide using metal chelating groups such as diethylenetriaminepentaacetic acid or ethylenediaminetetraacetic acid.

[0050] The marker can be a chemiluminescent compound, the presence of which is detected by measuring the luminescence produced during the chemical reaction. Examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromaticacridinium ester, imidazole, acridinium salts and oxalate. Similarly, bioluminescent compounds can be used for labeling oligonucleotides and are detected by measuring luminescence. In this case, the catalytic protein increases the efficiency of the chemiluminescent reaction. Examples of bioluminescent labeling compounds include luciferin, luciferase and aequorin.

[0051] As used herein and unless otherwise specified, the term "fluorescent dye" refers to a fluorescent compound that emits energy in the form of fluorescence when exposed to light. A "chromophore of a reporter dye" is a network of atoms of a reporter dye that, when exposed to light, emits radiation at a level detectable by conventional spectroscopic means.

[0052] Most fluorescent dyes (e.g., dyes containing sulfonate moieties) are essentially incompatible with the phosphoramidite chemistry used in solid phase oligonucleotide synthesis. This is mainly due to the instability of these dyes to the chemical conditions present in the deprotection of nucleic acids and the solid phase cleavage process, and strong bases (e.g., ammonia) or primary amines (e.g., methylamine) are most commonly used in the solid phase. For example, sulfonated fluorescent dyes such as Atto490LS (ATTO-TEC, Inc., Siegen, Germany), CF640R, CF680R (Bio-Techne, Inc., USA) and Dy380XL, as well as Dy395XL, Dy396XL (Dyomics, GmbH, Jena, Germany) and JA286 (Roche Molecular Systems, Pleasanton, CA) are incompatible with phosphoramidite-based DNA synthesis on solid phase supports.

[0053] As used herein and unless otherwise indicated, the term "non-fluorescent" refers to compounds that, when exposed to radiation, do not emit radiation at levels detectable by conventional spectroscopic means.

[0054] As used herein and unless otherwise indicated, the term "weakly fluorescent" refers to a compound that emits radiation at low levels detectable by conventional spectroscopic means when exposed to radiation.

[0055] As used herein and unless otherwise specified, the term "light" refers to electromagnetic energy having a wavelength that causes a reporter dye to fluoresce, wherein the wavelength may be in the range of 190-800 nm.

[0056] As used herein and unless otherwise indicated, the term "specificity" refers to a nucleic acid used in a reaction, such as a probe used in a hybridization reaction, a primer used in PCR, or a nucleic acid present in a composition, which, under normal detection conditions, hybridizes only to the intended target and not to other nucleic acid molecules in the test sample.

[0057] As used herein and unless otherwise indicated, the term "selective" refers to a nucleic acid used in a reaction, such as a probe used in a hybridization reaction, a primer used in PCR, or a nucleic acid present in a pharmaceutical preparation, that hybridizes to a target of interest more frequently, more rapidly, or for a longer period of time than it hybridizes to other nucleic acids in a test sample in a normal test environment.

[0058] As used herein, the term "hybridize under stringent conditions" describes conditions for hybridization and washing, under which nucleotide sequences that are at least 60% (65%, 70%, 75% or more) identical to each other generally remain hybridized to each other. Stringent conditions depend on the nature of the nucleic acid (e.g., length, GC content, etc.) and the method itself (hybridization, amplification, etc.). Such methods are well known in the art, see Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. In one embodiment, stringent hybridization conditions are hybridization at about 45°C in 6× sodium chloride / sodium citrate (SSC), followed by one or more washes at about 68°C in 0.1×SSC, 0.2% SDS. In another embodiment, stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. (i.e., one or more washes at 50° C., 55° C., 60° C., or 65° C.). It should be understood that the nucleic acids of the present invention do not include nucleic acid molecules that hybridize only to nucleotide sequences consisting of only A or T nucleotides under these conditions. For example, an oligonucleotide of about 15-40 bases can be stringently hybridized to a complementary sequence in a polymerase chain reaction (PCR) under the following conditions: a salt concentration of 50 mM KCl, a buffer concentration of 10 mM Tris·HCl, MgCl2, and a 10 mM sodium dodecyl sulfate. 2+ The concentration is 1.5 mM, the pH is 7-7.5, and the annealing temperature is 55-60°C. Moderately stringent hybridization of oligonucleotides of about 15-40 bases with complementary sequences in polymerase chain reaction (PCR) can be carried out under the following conditions: salt concentration is 50 mM KCl, buffer concentration is 10 mM Tris·HCl, Mg 2+ The concentration is 1.5 mM, the pH is 7-7.5, and the annealing temperature is 48-54°C. Low stringency hybridization of oligonucleotides of about 15-40 bases with complementary sequences in polymerase chain reaction (PCR) can be carried out under the following conditions: salt concentration is 50 mM KCl, buffer concentration is 10 mM Tris·HCl, Mg 2+ The concentration is 1.5 mM, the pH is 7-7.5, and the annealing temperature is 37-47°C.

[0059] As used herein and unless otherwise indicated, the term "stereoisomerically pure" refers to a composition comprising one stereoisomer of a compound and substantially free of other stereoisomers of the compound. For example, a stereoisomerically pure composition of a compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereoisomerically pure composition of a compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereoisomerically pure compound comprises greater than about 80% by weight of a stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, more preferably greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of other stereoisomers of the compound, even more preferably greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of other stereoisomers of the compound, most preferably greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of other stereoisomers of the compound.

[0060] As used herein and unless otherwise indicated, a "pure" or "substantially pure" compound refers to a composition that includes one compound and does not contain detectable or substantial amounts of other compounds. A typical substantially pure composition includes greater than about 80% by weight of the target compound and less than about 20% by weight of one or more other compounds, more preferably greater than about 90% by weight of the target compound and less than about 10% by weight of one or more other compounds, even more preferably greater than about 95% by weight of the target compound and less than about 5% by weight of one or more other compounds, and most preferably greater than about 97% by weight of the target compound and less than about 3% by weight of one or more other compounds.

[0061] The method of nucleic acid amplification is polymerase chain reaction (PCR), which is disclosed in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 and other references. PCR generally uses two or more oligonucleotide primers, which are combined with a selected nucleic acid template (e.g., DNA or RNA). Primers for nucleic acid analysis include oligonucleotides that can serve as a starting point for nucleic acid synthesis within the nucleic acid sequence of the target nucleic acid. Primers can be purified from restriction digests by conventional methods, or they can be synthesized. For maximum efficiency in amplification, primers can be single-stranded, but primers can be double-stranded. First, the double-stranded primer is denatured (i.e., treated) to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0062] If the template nucleic acid is double-stranded, the two strands must be separated before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturation method, including physical, chemical or enzymatic means. One method of separating nucleic acid strands involves heating the nucleic acid until it is mostly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing the template nucleic acid will depend on, for example, the buffer salt concentration and the length and nucleotide composition of the denatured nucleic acid, but typically range from about 90°C to about 105°C for a period of time depending on the reaction characteristics (such as temperature and nucleic acid length). Denaturation is typically performed for about 5 seconds to 9 minutes. In order not to expose the corresponding polymerase (e.g., Z05 DNA polymerase) to such high temperatures for too long and therefore be at risk of losing functional enzymes, a short denaturation step may preferably be used.

[0063] If the double-stranded template nucleic acid was denatured by heating, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the target nucleic acid.

[0064] The temperature for annealing can be about 35°C to about 70°C, or about 45°C to about 65°C; or about 50°C to about 60°C, or about 55°C to about 58°C. The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds; about 30 seconds to about 40 seconds). In this context, it can be advantageous to use different annealing temperatures to increase the inclusiveness of the corresponding assay. In short, this means that at relatively low annealing temperatures, primers can also bind to targets with a single mismatch, and therefore variants of certain sequences can also be amplified. This is also desirable if, for example, a certain organism has known or unknown genetic variants (which should also be detected). On the other hand, a relatively high annealing temperature has the advantage of providing higher specificity, because towards higher temperatures, the probability of primers binding to incompletely matched target sequences continues to decrease. In order to benefit from both phenomena, in some embodiments of the present invention, the above method includes annealing at different temperatures, such as first annealing at a lower temperature and then annealing at a higher temperature. If, for example, the first incubation is carried out at 55° C. for about 5 cycles, non-exactly matched target sequences can be (pre-)amplified. This can then be carried out, for example, at 58° C. for about 45 cycles, providing higher specificity throughout the main part of the experiment. In this way, potentially important genetic variants are not lost, while a high degree of specificity is maintained.

[0065] The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient to allow extension to occur from the annealed primers to generate a product complementary to the nucleic acid to be analyzed. The temperature should be sufficient to synthesize extension products from each primer annealed to the nucleic acid template, but should not be so high that the extension products are denatured from their complementary templates (e.g., the temperature for extension typically ranges from about 40°C to 80°C (e.g., about 50°C to about 70°C; about 65°C)). The extension time can be about 10 seconds to about 5 minutes, or about 15 seconds to 2 minutes, or about 20 seconds to about 1 minute, or about 25 seconds to about 35 seconds. The newly synthesized chain forms a double-stranded molecule that can be used in the subsequent steps of the reaction. The chain separation, annealing and extension steps can be repeated as many times as necessary to produce the desired number of amplification products corresponding to the target nucleic acid. The limiting factor in the reaction is the amount of primers, thermostable enzymes and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing and extension) can be repeated at least once. For use in detection, the number of cycling steps will depend, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence sufficient for detection. Typically, the cycling steps are repeated at least about 20 times, but can be repeated up to 40, 60 or even 100 times.

[0066] PCR can be performed in which the steps of annealing and extension are performed in the same step (one-step PCR) or, as described above, in separate steps (two-step PCR). Performing annealing and extension together, and therefore under the same physical and chemical conditions, with a suitable enzyme (such as, for example, Z05 DNA polymerase), has the advantage of saving time for an additional step in each cycle and also eliminating the need for additional temperature adjustments between annealing and extension. Thus, one-step PCR reduces the overall complexity of the corresponding assay.

[0067] Generally, a shorter time for overall amplification may be preferred because the time to result is reduced and leads to a possible earlier diagnosis.

[0068] Other nucleic acid amplification methods to be used include ligase chain reaction [LCR; Wu DY and Wallace RB, Genomics 4 (1989) 560-69; and Barany F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]; polymerase ligase chain reaction (Barany F., PCR Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO90 / 01069); repair chain reaction (EP 0439182 A2), 3SR (Kwoh DY et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; Guatelli JC et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92 / 08808), and NASBA (US 5,130,238). In addition, there are strand displacement amplification (SDA), transcription-mediated amplification (TMA) and Qb-amplification (for review, see, for example, Whelen AC and Persing DH, Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson RD and Myers TW, Curr. Opin. Biotechnol. 4 (1993) 41-47).

[0069] The term "Cp value" or "crossing point" value refers to a value that allows the input target nucleic acid to be quantified. The Cp value can be determined according to the second-derivative maximum method (Van Luu-The et al., "Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction", BioTechniques, Vol. 38, No. 2, February 2005, pp. 287-293). In the second-derivative method, Cp corresponds to the first peak of the second-derivative curve. This peak corresponds to the beginning of the logarithmic linear phase. The second-derivative method calculates the second-derivative value of the real-time fluorescence intensity curve, and only one value is obtained. The initial Cp method is based on a locally defined differentiable approximation of the intensity value, for example, by a polynomial function. Then the third-order derivative is calculated. The Cp value is the smallest root of the third-order derivative. The Cp can also be determined using the fitting point method, in which the Cp is determined by the intersection of parallel lines and threshold lines in the logarithmic linear region. The LightCycler instrument provided by Roche provides Cp values ​​by calculation according to the second derivative maximum method.

[0070] The term "PCR efficiency" refers to an indicator of cycle-to-cycle amplification efficiency. The PCR efficiency for each condition was calculated using the following equation: PCR efficiency % = (10 (-斜率) -1) x 100, where the slope is calculated by linear regression of the log copy number plotted on the y-axis and the Cp plotted on the x-axis. PCR efficiency can be measured using perfectly matched or mismatched primer templates.

[0071] The term "FRET" or "Fluorescence Resonance Energy Transfer" or " "Resonance Energy Transfer" refers to the transfer of energy between at least two chromophores, a donor chromophore and an acceptor chromophore (called a quencher). When the donor is excited by light radiation having a suitable wavelength, the donor typically transfers energy to the acceptor. The acceptor typically re-emits the transferred energy in the form of light radiation having a different wavelength. When the acceptor is a "dark" quencher, it dissipates the transferred energy in a form other than light. Whether a particular fluorophore acts as a donor or an acceptor depends on the properties of the other members of the FRET pair. Common donor-acceptor pairs include the FAM-TAMRA pair. Common quenchers are DABCYL and TAMRA. Common dark quenchers include BlackHoleQuenchers TM(BHQ) (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black TM (Integrated DNA Tech., Inc., Coralville, Iowa) and BlackBerry TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0072] The method described above can be based on FRET between a donor fluorescent moiety and an acceptor fluorescent moiety. A representative donor fluorescent moiety is fluorescein, and representative corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically, detection includes exciting the sample at a wavelength absorbed by the donor fluorescent moiety and developing and / or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. In the method according to the present invention, FRET can be quantitatively detected after detection. For example, detection is performed after each cycle step. For example, detection is performed in real time. By using a commercially available real-time PCR instrument (e.g., LightCycler TM or ), PCR amplification and detection of amplification products can be combined in a single closed cuvette with significantly reduced cycle times. Since detection occurs simultaneously with amplification, the real-time PCR method eliminates the need to manipulate the amplification products and reduces the risk of cross-contamination between amplification products. Real-time PCR greatly reduces turnaround time and is an attractive alternative to conventional PCR technology in clinical laboratories.

[0073] The following patent application describes Real-time PCR used in the techniques: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712. The instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high quality optics. This rapid thermal cycling technique uses a thin glass cuvette as the reaction vessel. The heating and cooling of the reaction chamber is controlled by alternating heated and ambient air. Due to the low mass of the air and the high surface area to volume ratio of the cuvette, very rapid temperature exchange rates can be achieved within the hot chamber.

[0074] Technology utilizes a single-stranded hybridization probe labeled by two fluorescent moieties. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the FRET principle. The second fluorescent moiety is generally a quencher molecule. Typical fluorescent dyes used in this form are, for example, especially FAM, HEX, Cy5, JA270, Cyan and Cy5.5. During the annealing step of the PCR reaction, the labeled hybridization probe is combined with the target nucleic acid (i.e., amplification product), and in the subsequent extension phase, it is degraded by 5' to 3' exonuclease activity of Taq or another suitable polymerase (such as mutant Z05 polymerase) as known to the technician. Therefore, the fluorescent moiety and the quencher moiety become spatially separated from each other. Therefore, after the first fluorescent moiety is excited in the absence of a quencher, the fluorescence emission from the first fluorescent moiety can be detected.

[0075] In both detection formats described above, the intensity of the emitted signal can be related to the number of original target nucleic acid molecules.

[0076] Recently, the use of “tagged” Method for performing multiplex PCR assays using probes.

[0077] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes [e.g., or (Molecular Probes, Inc., Eugene, OR)], the amplification product can be detected. When interacting with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after being excited with light of a suitable wavelength. Double-stranded DNA binding dyes (such as nucleic acid intercalating dyes) can also be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed to confirm the presence of the amplification product.

[0078] Molecular beacons conjugated to FRET can also be used to detect the presence of amplification products using the real-time PCR method of the present disclosure. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is usually a quencher, and the fluorescent label is generally located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow secondary structure formation (e.g., hairpins). As a result of the formation of the secondary structure in the probe, when the probe is in solution, the two fluorescent moieties are close in space. After hybridization with the amplification product, the secondary structure of the probe is destroyed, and the fluorescent moieties become separated from each other, so that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

[0079] Thus, the method according to the invention is a method using FRET as described above, wherein the probe comprises a nucleic acid sequence allowing secondary structure formation, wherein the secondary structure formation results in a spatial proximity between the first fluorescent moiety and the second fluorescent moiety.

[0080] Efficient FRET occurs only when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.

[0081] Thus, in one embodiment, the donor and acceptor fluorescent moieties are within no more than 5 nucleotides of each other on the probe. In a further embodiment, the acceptor fluorescent moiety is a quencher.

[0082] As mentioned above, in In the above format, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) during the annealing step of the PCR reaction and is degraded during the subsequent extension phase by the 5' to 3' exonuclease activity of Taq or another suitable polymerase known to the skilled person (such as mutant Z05 polymerase). Thus, in one embodiment, in the above method, amplification employs a polymerase with 5' to 3' exonuclease activity.

[0083] Further advantageously, the length of the amplicon produced by the above method is carefully selected. Generally, relatively short amplicon increases the efficiency of the amplification reaction. Thus, one aspect of the invention is the above method, wherein the amplified fragment comprises a maximum of 450 bases, a maximum of 300 bases, a maximum of 200 bases or a maximum of 150 bases.

[0084] A "sequence" is the primary structure of a nucleic acid, i.e., the specific arrangement of the individual nucleobases that make up each nucleic acid. It must be understood that the term "sequence" does not refer to a specific type of nucleic acid such as RNA or DNA, but applies to both as well as other types of nucleic acids, such as, for example, PNA or others. As is well known in the relevant art, where the nucleobases correspond to each other, particularly in the case of uracil (present in RNA) and thymine (present in DNA), these bases can be considered to be equivalent between RNA and DNA sequences.

[0085] Clinically relevant nucleic acid is often DNA, which can be derived from DNA viruses, such as, for example, hepatitis B virus (HBV), cytomegalovirus (CMV), etc., or bacteria, such as, for example, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), etc. In such a case, in order to reflect the target nucleic acid properties, it can be advantageous to use an internal control nucleic acid consisting of DNA. The terms "cell", "cell line" and "cell culture" can be used interchangeably, and all such names include offspring. Thus, the term "transformant" or "converted cell" includes the cells of primary transformation and the culture derived from the cell regardless of the number of transfer. Due to deliberate or unintentional mutations, all offspring may not be exactly the same in DNA content. The definition of transformant includes a mutant offspring with the same function as the function screened in the initially transformed cell. The cell can be prokaryotic or eukaryotic.

[0086] The term "control sequence" refers to a DNA sequence necessary for expressing an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, promoters, optional operator sequences, ribosome binding sites, forward and reverse regulatory elements (see U.S. Patent No. 4,666,848) and possible other sequences. It is known that eukaryotic cells utilize promoters, polyadenylation signals and enhancers.

[0087] The term "operably linked" refers to the positioning of a coding sequence so that a control sequence will function to drive the expression of a protein encoded by the coding sequence. Thus, a coding sequence "operably linked" to a control sequence represents a configuration in which the coding sequence can be expressed under the direction of the control sequence.

[0088] The terms "restriction endonuclease" and "restriction enzyme" refer to enzymes, usually of bacterial origin, that cleave double-stranded DNA at or near a specific nucleotide sequence.

[0089] Families of amino acid residues with similar side chains are defined herein. These families include amino acids with following side chains: basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, cysteine, glycine), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0090] The term "reagent solution" is any solution containing at least one reagent required or used for PCR purposes. Most typical ingredients are polymerase, nucleotides, primers, ions, magnesium, salts, pH buffers, nucleotide triphosphates (NTPs) or deoxynucleotide triphosphates (dNTPs), probes, fluorescent dyes (which can be attached to the probes), nucleic acid binding agents, nucleic acid templates. Reagents can also be additional polymerase reaction additives that have an impact on the polymerase reaction or its monitoring.

[0091] The term "master mix" refers to a mixture of all or most of the ingredients or factors required for PCR to occur, and in some cases, refers to all except the sample and amplicon-specific templates and primers. Commercially available master mixes are typically concentrated solutions. A master mix can contain all reagents common to a variety of samples, but it can also be constructed for only one sample. Using a master mix helps to reduce pipetting errors and variations between samples caused by differences in the volumes of pipetting.

[0092] The term "thermostable polymerase" refers to an enzyme that is heat-stable, heat-resistant, and retains sufficient activity to effect a subsequent primer extension reaction after being subjected to elevated temperatures for the time required for double-stranded nucleic acid denaturation. Typically, synthesis is initiated at the 3' end of each primer and proceeds in a 5' to 3' direction along the template strand. The heating conditions required for nucleic acid denaturation are well known in the art and are illustrated in U.S. Patent Nos. 4,965,188 and 4,889,818. As used herein, a thermostable polymerase is suitable for use in temperature cycling reactions such as PCR. Examples of thermostable nucleic acid polymerases include Thermus aquaticus Taq DNA polymerase, Thermus Z05 polymerase, Thermus flavivirida polymerase, Thermotoga maritima polymerases such as TMA-25 and TMA-30 polymerases, Tth DNA polymerase, and the like. However, non-thermostable polymerases can also be used in PCR assays provided that the enzyme is supplemented.

[0093] A "polymerase with reverse transcriptase activity" is a nucleic acid polymerase that can synthesize DNA based on an RNA template. Once RNA has been reverse transcribed into single-stranded cDNA, it can also replicate single-stranded or double-stranded DNA. In one embodiment of the invention, the polymerase with reverse transcriptase activity is thermostable.

[0094] In RNA molecule amplification by DNA polymerase, the first extension reaction is reverse transcription using the RNA template, and a DNA strand is produced. The second extension reaction using the DNA template produces a double-stranded DNA molecule. Therefore, synthesis of a complementary DNA strand from the RNA template by DNA polymerase provides raw materials for amplification.

[0095] Thermostable DNA polymerase can be used in coupled single enzyme reverse transcription / amplification reactions. In this context, the term "homogeneous" refers to a two-step single addition reaction for reverse transcription and amplification of an RNA target. Homogeneity means that after the reverse transcription (RT) step, there is no need to open the reaction vessel or otherwise adjust the reaction components before the amplification step. In non-homogeneous RT / PCR reactions, after reverse transcription and before amplification, for example, one or more reaction components (such as an amplification reagent) are adjusted, added or diluted, and the reaction vessel must be opened for this, or at least its content must be operated. The scope of the present invention includes both homogeneous and non-homogeneous embodiments.

[0096] Reverse transcription is the basic step in RT-PCR.For example, RNA templates known in the art show a tendency to form a secondary structure, which can hinder primer binding and / or cDNA chain extension by corresponding reverse transcriptase.Thus, the relatively high temperature of RT reaction is favourable with regard to the efficiency of transcription.On the other hand, improving the incubation temperature also means higher specificity, i.e. RT primers will not anneal to the sequence showing the mispairing with one or more sequences expected.Particularly in the case of a plurality of different target RNAs, it can be expected that it also transcribes and subsequently increases and detects a sequence with a single mispairing, for example, in the case of a substrain or subspecies of an unknown or rare organism that may be present in a fluid sample.

[0097] In order to benefit from the two advantages mentioned above, namely the reduction of secondary structure and reverse transcription of templates with mismatches, the RT incubation can be performed at more than one different temperature.

[0098] Therefore, one aspect of the present invention is the method described above, wherein said incubation of a polymerase having reverse transcriptase activity is carried out at a temperature varying from 30°C to 75°C, or from 45°C to 70°C, or from 55°C to 65°C.

[0099] As a further important aspect of reverse transcription, a long RT step can damage DNA templates that may be present in the fluid sample. If the fluid sample contains both RNA and DNA species, it is therefore advantageous to keep the duration of the RT step as short as possible, but at the same time ensure that sufficient amounts of cDNA are synthesized for subsequent amplification and optional detection of the amplification products.

[0100] Thus, one aspect of the invention is the method described above, wherein the time period for incubating the polymerase having reverse transcriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes or 1 minute.

[0101] A further aspect of the invention is the method as described above, wherein the polymerase having reverse transcriptase activity and comprising a mutation is selected from:

[0102] a) CS5 DNA polymerase;

[0103] b) CS6 DNA polymerase;

[0104] c) Thermotoga maritima DNA polymerase;

[0105] d) Thermus aquaticus DNA polymerase;

[0106] e) Thermus thermophilus DNA polymerase;

[0107] f) Thermus flavus DNA polymerase;

[0108] g) Thermus filiformis DNA polymerase;

[0109] h) Thermus sp. sps17 DNA polymerase;

[0110] i) Thermus sp. Z05 DNA polymerase;

[0111] j) Thermotoga neapolitana DNA polymerase;

[0112] k) Termosipho africanus DNA polymerase;

[0113] l) Thermus caldophilus DNA polymerase.

[0114] Particularly suitable for these requirements are enzymes carrying mutations in the polymerase domain that enhance their reverse transcription efficiency in terms of faster elongation rates.

[0115] Thus, in the above methods, the polymerase having reverse transcriptase activity is an enzyme comprising a mutation that confers an improved nucleic acid extension rate and / or improved reverse transcriptase activity relative to a corresponding wild-type polymerase.

[0116] In one embodiment, in the above method, the polymerase having reverse transcriptase activity is an enzyme comprising a mutation that confers improved reverse transcriptase activity relative to a corresponding wild-type polymerase.

[0117] WO 2008 / 046612 discloses polymerases carrying point mutations that make them particularly useful. Specifically, the polymerase to be used may be a mutant DNA polymerase comprising at least the following motif in the polymerase domain:

[0118] TGRLSS-Xb7-Xb8-PNLQN; wherein Xb7 is an amino acid selected from S or T, and wherein Xb8 is an amino acid selected from G, T, R, K or L, wherein the polymerase comprises 3′ to 5′ exonuclease activity and has an improved nucleic acid extension rate and / or improved reverse transcription efficiency relative to a wild-type DNA polymerase, wherein in the wild-type DNA polymerase, Xb8 is an amino acid selected from D, E or N.

[0119] An example is a mutant of a thermostable DNA polymerase from Thermus sp. Z05 (described, for example, in US 5,455,170), the variant comprising a mutation in the polymerase domain compared to the corresponding wild-type enzyme Z05. An embodiment of the method according to the invention is a mutant Z05 DNA polymerase, wherein the amino acid at position 580 is selected from the group consisting of G, T, R, K and L. Other examples of mutant Z05 DNA polymerases with improved reverse transcription efficiency include, but are not limited to, the D580G, E522G mutants described in US9,090,883; the D580G, I709K mutants described in US9,017,979; the D580G, I709K, I640F mutants described in US8,759,063; the D580G, I709K, I616M mutants described in US8,945,882; and the D580G, I709K, Y698F mutants described in US9,080,156.

[0120] For reverse transcription using thermostable polymerases, divalent cations (such as Mn) are typically included in the form of a salt. 2+ or Mg 2+ ), such as manganese chloride (MnCl2), manganese acetate [Mn(OAc)2] or manganese sulfate (MnSO4) or magnesium chloride (MgCl2), magnesium acetate [Mg(OAc)2] or magnesium sulfate (MgSO4). If MnCl2 is included in a reaction containing 50 mM Tricine buffer, for example, MnCl2 is typically present at a concentration of 0.5-7.0 mM; when 200 μM each of dGTP, dATP, dUTP and dCTP are used, 2.5-3.5 mM is typically present.

[0121] A "modified" thermostable polymerase refers to a polymerase in which at least one monomer is different from a reference sequence, such as a native or wild-type form of a polymerase or another modified form of a polymerase. Exemplary modifications include monomer insertions, deletions, and substitutions. Modified polymerases also include chimeric polymerases having identifiable component sequences (e.g., structural or functional domains, etc.) derived from two or more parents. Also included within the definition of modified polymerases are those that contain chemical modifications of the reference sequence. Further examples of modified thermostable polymerases include G46E E678G CS5 DNA polymerase, G46E L329A E678G CS5 DNA polymerase, G46E L329A D640G S671F CS5 DNA polymerase, G46EL329A D640G S671F E678G CS5 DNA polymerase, G46E E678G CS6 DNA polymerase, Z05 DNA polymerase, ΔZ05 polymerase, ΔZ05-Gold polymerase, ΔZ05R polymerase, E615G Taq DNA polymerase, E678G TMA-25 polymerase, E678G TMA-30 polymerase, and the like.

[0122] The term "thermoactive polymerase" refers to an enzyme that is active at elevated temperatures (eg, 55-80°C) necessary to ensure specific priming and primer extension.

[0123] The terms "peptide," "polypeptide," and "protein" are used interchangeably. The terms "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably. Unless otherwise indicated, amino acid sequences are written from the amino terminus to the carboxyl terminus. Single-stranded nucleic acid sequences are written from 5' to 3' unless otherwise indicated. Double-stranded nucleic acid sequences are written from 5' to 3' for the top strand, and from 3' to 5' for the bottom strand, unless otherwise indicated.

[0124] The following examples are given to illustrate embodiments of the invention as it is presently preferably practiced. It should be understood that the examples are illustrative and that the invention should not be considered to be limiting except as indicated in the appended claims.

[0125] Examples

[0126] Abbreviations: dATP = 2′-deoxyadenosine 5′-triphosphate, dCTP = 2′-deoxycytidine 5′-triphosphate, DEPC = diethyl pyrocarbonate, dGTP = 2′-deoxyguanosine 5′-triphosphate, DIPEA = N,N-diisopropylethylamine, DMF = N,N′-dimethylformamide, DMSO = dimethyl sulfoxide, DMT-MM = 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt, dUTP = 2′-deoxyuridine 5′-triphosphate, eq. = equivalent, HEPES = 4′-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholinium salt. BS = N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid), HEPPS = 4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid, LNA = locked nucleic acid, MeCN = acetonitrile, NHS = N-hydroxysuccinimide, NMP = 1-methyl-2-pyrrolidone, qPCR = real-time polymerase chain reaction, RT = room temperature, TEAA = triethylammonium acetate, TEAB = triethylammonium bicarbonate, Tris·HCl = tris(hydroxymethyl)aminomethane hydrochloride, UPLC-MS = ultra-performance liquid chromatography coupled to mass spectrometry.

[0127] General Materials and Methods: DNA oligonucleotides and DNA / LNA hybrid oligonucleotides with primary amino modifications were synthesized by solid phase DNA synthesis using amino modifier phosphoramidites. For 5′-terminal modification, 6-(trifluoroacetamido)-hexyl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite was used. For internal modifications, 5′-dimethoxytrityl-5-[N-(trifluoroacetamidohexyl)-3-acrylimide]-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite were used. If BHQ-2 was located at the 5′-terminus, all DNA sequences contained 3′- or 5′-BHQ-2 (Black Hole ) and 3′-phosphoric acid. Water was ultra-high purity water with a resistivity of at least 18.2 MΩcm at 25°C. All components of quantitative PCR (qPCR) were prepared with DEPC-treated water.

[0128] Example 1: Labeling DNA with fluorescent dyes

[0129] DNA pretreatment: Unpurified DNA oligonucleotides (crude) obtained from standard solid phase DNA synthesis were deprotected and desalted using standard methods to remove any traces of residual amines. Oligonucleotides (1 μmol of synthesis scale) were lyophilized and redissolved in water (100 μL) with the aid of a warm water bath. Lithium chloride aqueous solution (80 μL, 10M) was added, and the mixture was shaken vigorously for 10 min. Ice-cold ethanol (3.3 mL) was added, and the test tube was gently inverted several times. The mixture was cooled at -20 ° C for 20 min and centrifuged (2500 rpm) at 7 ° C for 30 min in a precooled centrifuge. The colorless supernatant was carefully decanted and discarded. The remaining solid was washed with anhydrous ethanol, dried under high vacuum, and redissolved in an aqueous buffer (1 mL, 50 mM, pH 8.0-9.0). The buffer was selected from borate, carbonate, HEPBS, HEPPS or TEAB. The amount of DNA was analyzed by UPLC to determine the amount of amino-modified target sequence. Determine the total nucleic acid concentration using a spectrophotometer using the calculated extinction coefficient of the target DNA sequence at 260 nm.

[0130] Pre-activation: fluorescent dyes Atto490LS (ATTO-TEC, Inc., Siegen, Germany), CF640R, CF680R, (Bio-Techne, Inc., USA), Chromeo TM 494 (Active Motif, Inc., Carlsbad, CA), Coumarin 343 (C343), Dy380XL, Dy395XL, Dy396XL (Dyomics GmbH, Jena, Germany) and JA286 (Roche Molecular Systems, Pleasanton, CA) were obtained as carboxylic acids and used without further manipulation. In a glass vial, the fluorescent dye (1.0 equivalent of carboxylic acid) was dissolved in a dry organic solvent (20 mM). Depending on the solubility of the marker, the solvent was selected from DMSO, NMP, DMF or a 50% mixture thereof with water. For labeling reactions requiring less than 1 mg of carboxylic acid, the concentration was adjusted by absorption spectroscopy using the extinction coefficient of the chromophore. DIPEA (1.2 equivalents) or another weak non-nucleophilic base was added and the solution was briefly mixed. In a separate reaction vial, the tetrafluoroborate salt of DMT-MM (2.0 equivalents) was weighed, the carboxylic acid solution was added, and then vigorously mixed until all solids were dissolved. Preactivation was performed on a Thermoshaker at RT for 15 min.

[0131] Labeling reaction: The pre-activated solution (75 μL, 3.0 eq.) was quickly mixed with the DNA solution (100 μL, 1.0 eq. primary amine) and the labeling reaction was performed on a Thermoshaker at RT. The progress of the reaction was monitored by UPLC analysis, and a sample of the reaction mixture (1 μL) was diluted with water (19 μL) before injection into the UPLC (7 μL). When UPLC analysis indicated that the reaction was complete, the solution was diluted ten-fold with TEAA buffer (100 mM, pH 7.0) or Tris·HCl buffer (50 mM, pH 7.5) and stored frozen until purification. The results of the UPLC analysis are shown in Figures 2-5.

[0132] Purification: The labeled DNA was purified by reverse phase liquid chromatography using Tris·HCl (50 mM, pH 7.5) or TEAA (0.1 mM, pH 7.0) and MeCN. Figure 6 As shown. The combined product fractions were concentrated on a centrifugal vacuum concentrator and desalted by size exclusion chromatography. The purified DNA probe (0.1 mM) was lyophilized and then redissolved in TE buffer (10 mM Tris·HCl, 1 mM EDTA) for qPCR.

[0133] Example 2: qPCR performance of DNA probes

[0134] By directly comparing the qPCR performance of probes prepared using DMT-MM reagents with those prepared using traditional NHS ester chemistry, questions about the quality and functionality of DNA probes can be addressed. The target DNA sequences are based on the pathogens Trichomonas vaginalis and Mycoplasma genitalium.

[0135] Prepare the following qPCR mix in multiple volumes for single replicate reactions. The master mix contains trioctylamine buffer (30 mM, pH 8.7), potassium acetate (40 mM), DMSO [1.08% (v / v)], EDTA (20 μM), 20 [0.02% (w / v)], sodium azide [0.09% (w / v)], dATP (0.4 mM), dCTP (0.4 mM), dGTP (0.4 mM) and dUTP (0.4 mM), forward and reverse primers (1 μM each) and DNA probe (0.1 μM). The cofactor mixture contained magnesium sulfate (1 M) and sodium azide [10% (w / v)]. The enzyme mixture contained trioctylamine buffer (0.28 M, pH 8.75), potassium acetate (0.28 M), sodium azide [0.09% (w / v)], EDTA (155 μM), DMSO [7.56% (v / v)], glycerol [7.015% (v / v)], aptamer NTQ-46A (2.8 μM), polymerase (5.4 U / μL) and uracil DNA glycosylase (1.3 U / μL). The elution buffer contained Tris·HCl, bovine serum albumin [1.25 mg / mL (w / v)] and sodium azide [0.09% (w / v)].

[0136] The sample tubes were filled with master mix (10 μL, fragment 10), cofactor mix (10 μL, fragment 9), enzyme mix (10 μL, fragment 8), and elution buffer containing qPCR (40 μL, fragment 7). Target DNA was added to fragment 7. qPCR was performed for five cycles with denaturation at (95°C, 3s), annealing at (61°C, 1s), and extension at (62°C, 3s). Continuous cycles were performed with denaturation at (93°C, 2s), annealing at (61°C, 1s), and extension at (62°C, 3s). For each DNA probe, five cycles were performed. Five replicates were run on the system unit.

[0137] Results: Figure 7 and 8 As shown, DNA probes labeled by in situ activation with DMT-MM showed higher baseline fluorescence intensity and improved K exp The coefficient of qPCR was 0.1, indicating that qPCR was more reliable. In conclusion, DNA probes prepared by in situ activation with DMT-MM showed comparable or better performance.

[0138] Example 3: Large-scale labeling of DNA with fluorescent dyes

[0139] DNA oligonucleotide starting materials were obtained from standard solid phase synthesis on a 15 μmol scale and used in labeling reactions without purification.

[0140] DNA pretreatment: The crude DNA oligonucleotides were desalted, lyophilized, and then redissolved in water (1 mL) with the aid of a warm water bath. Dry sodium iodide was added to a final concentration of 10 M, ensuring that the salt was completely dissolved. The mixture was diluted with ethanol (200 standard solution, 30 mL), vortexed briefly, and then kept at room temperature (RT) for 15 min. The precipitate formed was separated by centrifugation at RT (4000 rpm, 5 min). After pouring off the supernatant, the solid precipitate was thoroughly washed with ethanol (2×10 mL) and then dried under high vacuum. For the labeling reaction, the DNA oligonucleotides were redissolved in TEAB buffer (0.1 M, pH 8.5) and the analytical amount was analyzed by UPLC to determine the amount of amino-modified target sequence. The total nucleic acid concentration was determined using a spectrophotometer using the calculated extinction coefficient of the target sequence at 260 nm.

[0141] Pre-activation: Fluorescent dye (1.0 equivalent of carboxylic acid) was dissolved in dry DMSO (6 mM). DIPEA (1.1 equivalent) was added and the solution was briefly mixed. In a glass vial, DMT-MM tetrafluoroborate (2.0 equivalent) was weighed and immediately added to the carboxylic acid solution, followed by mixing until all solids were dissolved. Pre-activation was performed on a Thermoshaker at RT for 15 min.

[0142] Labeling reaction: The pre-activation solution (3.0 equivalents of carboxylic acid) was quickly added to a stirred solution of DNA raw material (1.0 equivalents of primary amine) in a conical reaction bottle. The concentration of organic solvent was 40%, and the final DNA concentration of the target sequence was between 1.5 and 1.7 mM, depending on the purity of the crude DNA. The labeling reaction was stirred at RT for 20 min, analyzed by UPLC, and diluted tenfold with TEAA buffer (100 mM, pH 7.0) for subsequent purification.

[0143] Purification: The labeled DNA was purified by reverse phase liquid chromatography using TEAA (0.1 mM, pH 7.0) and MeCN. The combined product fractions were purified by solid phase extraction (C 18 ) were desalted, lyophilized to dryness, and then redissolved in TE buffer (10 mM Tris·HCl, 1 mM EDTA) for qPCR.

[0144] Results: The labeling efficiency of two representative large-scale labeling reactions was measured using UPLC, each using a different synthesis batch of the fluorescent dye (Dy396XL), as shown in Figure 2. Fig.9A and 9B The purity and integrity of the purified products were determined by UPLC-MS. The final purity analysis of the DNA probes obtained from the six large-scale labeling reactions and their measured molecular weights are shown in Fig. 10A and Fig. 10BAs shown. The labeled DNA probe was subjected to functional analysis as described in Example 2. Fig.11 shown.

Claims

1. A method for post-synthetic modification of a nucleic acid using a detectable label, the method comprising the following steps: (a) preparing an amino-modified nucleic acid molecule comprising one or more amino modifications; (b) activating the carboxyl-modified label using a 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMT-MM) cation in the presence of a counter anion that is a weakly coordinating or non-coordinating anion, wherein the weakly coordinating or non-coordinating anion is not a carboxylic acid; and (c) reacting the amino-modified nucleic acid molecule with an activated carboxyl-modified label to produce a labeled nucleic acid molecule, wherein the detectable label is selected from the group consisting of a luminescent dye, a quencher dye and an affinity tag; and wherein the weakly coordinating or non-coordinating anion is selected from the group consisting of tetrafluoroborate (BF4 - ), hexafluorophosphate (PF6 - ), benzenesulfonate (C6H5SO3 - ), bis(trifluoromethanesulfonyl)imide((N[SO2(CF3)]2) - ), bromide ion (Br - ), 10-camphorsulfonate, iodide ion (I - ), methanesulfonate (methanesulfonate, CH3SO3 - ), perchlorate (ClO4 - ), phosphate (PO4 3- ), sulfate (SO4 2- ), tetrachloroaluminate (AlCl4 - ), tetrakis[3,5-bis(trifluoro-methyl)phenyl]borate (B[3,5-(CF3)2C6H3]4 - ), tetrakis(hexafluoroisopropyl)aluminate (Al[OC(CF3)3]4 - ), tetrakis(pentafluorophenyl)borate [B(C6F5)4 - ], p-toluenesulfonate (toluenesulfonate, CH3C6H4SO3 - ) and trifluoromethanesulfonate (trifluoromethanesulfonate, CF3SO3 - ).

2. The method according to claim 1, wherein the weakly coordinating or non-coordinating anion is tetrafluoroborate (BF4 - ) or hexafluorophosphate (PF6 - ). The method according to claim 1 , wherein the luminescent dye is a fluorescent dye or a phosphorescent dye.

4. The method according to any one of claims 1 to 3, wherein the nucleic acid molecule is an oligonucleotide. The method according to claim 4 , wherein the oligonucleotide is amino-modified at the 5′ end or the 3′ end. The method of claim 4 , wherein the oligonucleotide is internally amino-modified.

7. The method according to any one of claims 4 to 6, wherein the oligonucleotide is synthesized using phosphoramidite-based DNA synthesis.

8. The method of claim 7, wherein the oligonucleotide is purified after synthesis.

9. The method of claim 7, wherein the oligonucleotide is not purified after synthesis.

10. The method according to any one of claims 1 to 9, wherein the label is a fluorescent dye.

11. The method of claim 10, wherein the fluorescent dye is intrinsically incompatible with phosphoramidite-based DNA synthesis.

12. The method of any one of claims 3 to 11, wherein the fluorescent dye comprises a sulfonate moiety.

13. The method according to any one of claims 1 to 12, wherein said steps are performed during oligonucleotide synthesis, and wherein said oligonucleotide is bound to a solid support.

14. The method according to any one of claims 1 to 13, wherein the nucleic acid molecule is a product of enzymatic amplification.

15. The method according to any one of claims 1 to 14, wherein the nucleic acid molecule is single-stranded.

16. The method according to any one of claims 1 to 14, wherein the nucleic acid molecule is double-stranded.

17. The method according to any one of claims 1 to 15, wherein the nucleic acid molecule is selected from the group consisting of DNA, RNA, locked nucleic acid (LNA), peptide nucleic acid (PNA), nucleic acid analogs, and hybrid mixtures of DNA, RNA, LNA, PNA and nucleic acid analogs.

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