CN113980050B - A modified nucleotide, composition and reagent - Google Patents

Abstract

The present invention relates to the field of biological detection, in particular to a modified nucleotide, a composition and a reagent. The modified nucleotide disclosed in the present invention has only one position of substitution, which is convenient for synthesis; and the modified acyCTP is applied to mass spectrometry In the detection of SNP, while ensuring the elongation efficiency of single base elongase, the product molecular weight can be distinguished, the resolution of mass spectrum can be improved, and the accuracy of result interpretation can be ensured. The invention discloses a new idea of nucleic acid mass spectrometry detection. Detection is of great significance.

Description

A modified nucleotide, composition and reagent

Field of the Invention

The present invention relates to the field of biological detection, and in particular to a modified nucleotide, a composition and a reagent.

Background of the Invention

Single nucleotide polymorphism (SNP) refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genomic level. The frequency of occurrence in the population is higher than 1%, which is the most common type of human heritable variation. Therefore, the detection of SNP plays an extremely important guiding role in the diagnosis, screening and medication of genetic diseases.

At present, the methodologies for SNP detection mainly include real-time fluorescence PCR, PCR gene chip, PCR electrophoresis, PCR capillary electrophoresis analysis, PCR high-resolution melting curve, flow fluorescence hybridization, time-of-flight mass spectrometry, pyrophosphate sequencing, Sanger sequencing, etc. Among them, the advantages of real-time fluorescence PCR, PCR electrophoresis, PCR capillary electrophoresis analysis, PCR high-resolution melting curve and flow fluorescence hybridization are that they are time-consuming and highly sensitive, and can achieve detection in certain scenarios. However, due to their limited throughput, they cannot conveniently and quickly meet the clinical detection needs for dozens or even hundreds of gene sites. Although the gene chip method, pyrophosphate sequencing and Sanger sequencing are more accurate in detection, they are more expensive and time-consuming, and are not the first choice for SNP detection. Time-of-flight mass spectrometry has fast detection speed, simple data analysis and high throughput, which makes up for the shortcomings of traditional methodology and reduces costs. Compared with the previous methods, it is a better choice, but there is a problem of inaccurate detection results, which limits the further development of its application.

Nucleic acid mass spectrometry SNP detection is mainly based on PCR and primer extension technology. Its principle is to first amplify the target fragment of the SNP site to be detected by PCR primers, and the generated PCR products are treated with shrimp alkaline phosphatase (SAP) to neutralize the residual dNTPs. After the SAP digestion reaction is completed, buffer, extension primers, dideoxynucleotide (ddNTPs) and single base extension enzymes are added to the reaction solution for primer extension reaction. With the DNA amplification product as a template, the extension primer can bind to the 5' end of the SNP site to be detected and extend one base. After the primer extension is completed, the positive value is added to the reaction solution for desalting treatment to remove the metal ions adsorbed on the nucleic acid fragment. After desalting is completed, the sample and the matrix are transferred to the target plate to form a co-crystallization, and the spectrum is obtained by mass spectrometry detection. The typing of the SNP site of the sample can be analyzed by calculating the difference between the molecular weight of the product in the spectrum and the molecular weight of the extension primer.

However, when ddNTP is used as a substrate, the binding efficiency of the single-base extension enzyme is low and the extension effect is poor, which affects the accuracy of the spectrum result judgment. Another nucleotide substrate that can be used for single-base extension is linear nucleotides (acyclonucleotides, acyNTP), which replaces the common 2′-deoxyribofuranosyl sugar in dNTP with 2-hydroxyethoxymethyl group (as shown below). The recognition efficiency of DNA polymerase for acyNTP is 30 times that of ordinary ddNTP (reference: Gardner, A., and Jack, W. (2002). Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases. Nucleic Acids Res. 30, 605–613. doi: 10.1093/nar/30.2.605).

Summary of the invention

In order to solve the problem of inaccurate detection results caused by poor extension effect in nucleic acid mass spectrometry SNP detection, the present invention uses the principle that molecular weight difference can be used to analyze SNP site typing, and discloses a modified nucleotide, the structure of which is as follows:

wherein R is selected from alkyl; cycloalkyl; -OR ₁ ; -SR ₁ ; -SO ₃ H; -NR ₁ R ₂ and halogen; optionally substituted aryl or heterocyclic group; n is an integer from 1 to 12;

A is selected from _CH2 or O;

The molecular weight of the nucleotides is 474-924 Da.

Preferably, the molecular weight of the nucleotide is 481-924 Da.

In the prior art, acyNTP is mainly used as a substrate for sequencing, and is rarely used in nucleic acid mass spectrometry SNP detection. When acyNTP is used as a substrate, the molecular weights of acyCTP, acyATP, acyGTP and acyTTP are 425.12Da, 449.12Da, 465.14Da and 440.10Da, respectively.

It can be seen that the molecular weights of acyATP and acyTTP are close to each other. The difference in molecular weights between nucleotides determines that the nucleic acid mass spectrometry SNP detection project needs to distinguish peak signals with a difference of 9Da. However, it is difficult for a mass spectrometer to effectively distinguish characteristic peaks with a difference of 9Da, especially in the large molecular weight detection range such as 7000Da-12000Da, which results in the inability to accurately distinguish some SNP types. Only a mass spectrometer with particularly good resolution can achieve 9Da resolution, while ordinary mass spectrometers usually require peak signals with a molecular weight of more than 9Da to be separated. Characteristic peaks that are closely spaced will partially overlap, resulting in difficulty in resolution by mass spectrometers. When the molecular weight differs by 16Da, even on an ordinary mass spectrometer, the peak signals are separated far enough to be easily and accurately distinguished.

Therefore, the present invention uses acyUTP instead of conventional acyTTP, which increases the molecular weight difference between acyATP and acyTTP. The performance of acyUTP is close to that of acyTTP, and both can perform base complementary pairing with A.

The molecular weights of acyCTP, acyATP, acyGTP and acyUTP are 425.12Da, 449.12Da, 465.14Da and 426.10Da respectively.

The characteristic peaks of acyCTP and acyUTP almost overlap and cannot be distinguished. In actual operation, you can choose to change the molecular weight of one of them to achieve the purpose of distinguishing the characteristic peaks.

The present invention selects to modify acyCTP to change its molecular weight, and selects nucleotides with a molecular weight of more than 474Da, preferably more than 481Da, to ensure that the single-base extension enzyme efficiently recognizes the nucleotide substrate, while allowing the molecular weights of each nucleotide to be distinguished, thereby improving the spectrum resolution and the accuracy of result interpretation.

Preferably, the R is selected from C ₁ -C ₁₀ alkyl, more preferably C ₄ -C ₁₀ alkyl;

Preferably, the alkyl group may be substituted, and a preferred substituent is -NH ₂ .

Preferably, R ₁ and R ₂ are independently selected from H or C ₁ -C ₂₀ alkyl, preferably C ₁ -C ₁₀ alkyl, and more preferably C ₃ -C ₁₀ alkyl; preferably, halogen is selected from: F, Cl, Br or I, and more preferably Cl, Br or I;

Preferably, the heterocyclic group is selected from saturated heterocyclic groups, preferably 5-8 membered saturated heterocyclic groups; for example: tetrahydrofuranyl, morpholinyl, piperidinyl, piperazinyl; or the heterocyclic ring is selected from heteroaryl, preferably 5-10 membered heteroaryl, for example furanyl, pyrrolyl, thienyl, pyrazolyl, imidazolyl, oxazolyl, thiazolyl, pyridinyl, pyranyl, pyridazinyl, pyrimidinyl, pyrazinyl. The heterocyclic group is independently optionally substituted by one or more C ₁ -C ₁₀ alkyl groups; halogen, -OR ₁ or -NR ₁ R ₂ .

Preferably, illustrative, non-limiting specific examples of the compounds of formula I of the present invention are as follows:

In another aspect, the present invention discloses a substrate mixture, wherein the substrate mixture comprises Formula 1.

Preferably, the substrate mixture further comprises acyATP, acyGTP and acy UTP, and the structural formulas are as follows:

In another aspect, the present invention discloses a reagent for primer extension, comprising the above substrate mixture.

On the other hand, the present invention discloses a kit for nucleic acid mass spectrometry detection, comprising the above-mentioned reagent for primer extension.

Beneficial effects:

1. The modified nucleotide disclosed in the present invention has only one substitution position, which is convenient for synthesis.

2. The present invention creatively applies the modified acyCTP to the detection of mass spectrometry SNPs, which ensures the extension efficiency of the single-base extension enzyme while distinguishing the molecular weight of the products, improving the mass spectrometry resolution, and ensuring the accuracy of the result interpretation.

Description of the drawings:

Figure 1: The results of using acysulfo-CTP as a substrate to detect the mutation site 281C＞T;

Figure 2: Mass spectrum of clinical detection of acy sulfo-CTP as substrate;

Figure 3: Result of using acybutyl ether-CTP as substrate to detect mutation site IVS-I-5 (G>C);

Figure 4: Detection effect diagram of 6 nucleotides.

Specific implementation method:

Preparation Example: Modified Synthesis of Nucleotides

The amounts of all added substances are in excess relative to compounds I, II and III. The difference in the synthesis of different substances lies in the different amounts of products obtained.

A: When A is O, n = 1

(1) Synthesis of Compound II

Weigh 30 mmol of compound 1 and add it to a clean 250 mL three-necked flask; add 150 mL of dichloromethane and replace with nitrogen three times; protect with nitrogen; the system is heterogeneous; weigh 50 mmol of acyclovir side chain and add it to the above three-necked flask at one time; the system is heterogeneous; measure 7.5 mL of N,O-bistrimethylsilylacetamide and add it to the above three-necked flask at one time; the above reaction mixture is stirred at room temperature overnight; the system is still heterogeneous; measure 4 mL N,O-bistrimethylsilyl acetamide was added to the three-necked flask at one time; the reaction mixture was stirred at room temperature for 3 hours again, and the system became clear; the reaction system was cooled to 0°C with an ice-water bath; 1.2 mL of anhydrous tin tetrachloride was measured and added to the three-necked flask at one time; the reaction mixture was naturally heated to room temperature without removing the cold bath, and then stirred at room temperature overnight; the reaction mixture was carefully poured into 250 mL of saturated sodium bicarbonate aqueous solution, extracted with 250 mL of dichloromethane for 3 times, and the organic phases were combined and dried with anhydrous sodium sulfate and spin-dried; the residual light sulfonic solid was slurried with 200 mL of methyl tert-butyl ether to obtain a white solid;

(2) Synthesis of Compound III

The compound II synthesized in (1) was added to a clean 250 mL single-necked flask and dissolved with 100 mL of methanol; the atmosphere was replaced with nitrogen three times and then protected with nitrogen; 5.2 mmol of sodium tert-butoxide was weighed with weighing paper and added to the single-necked flask at one time; the reaction mixture was stirred at room temperature overnight and then directly dried by spin drying. The residue was dispersed in 50 mL of ethyl acetate, acidified with 1N hydrochloric acid, extracted with 50 mL of ethyl acetate four times, and the organic phases were combined and dried over anhydrous sodium sulfate to obtain a white solid by spin drying.

(3) Synthesis of Compound IV

Weigh 0.2mmol of compound III and add it to a clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace with nitrogen three times for nitrogen protection; weigh 0.65mmol of dried pyridine and add it to the above eggplant-shaped bottle at one time; cool the reaction system to 0°C with an ice-water bath; weigh 0.3mmol of phosphorus oxychloride and add it dropwise to the above eggplant-shaped bottle; without removing the cooling bath, stir the above reaction mixture at 0°C for 30 minutes and use it as solution A for later use; weigh 0.55mmol of tri-n-butylamine pyrophosphate and add it to another clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace with nitrogen Change three times, nitrogen protection; measure 1.5mL of dried tri-n-butylamine, add it to the above-mentioned eggplant-shaped bottle at one time; cool the reaction system to 0℃ with an ice-water bath; add solution A dropwise to the above-mentioned reaction system; without removing the cooling bath, stir the above-mentioned reaction mixture at 0℃ for 30 minutes; measure 4mL of deionized water, add it to the above-mentioned reaction system at one time, and stir at room temperature for 2 hours; after concentrating the organic solvent at 0℃, the residual aqueous solution is separated and purified by a high-pressure preparative separation and purification system (Pre-HPLC); the obtained eluate is lyophilized to obtain a white solid, which is redissolved in 1mL of cold deionized water and frozen at -20℃.

Among them, the different substitutions of X, compound IV can be verified by listing the following structures.

When n=1, the molecular weight is as shown in Table 1.

Table 1 Compounds and mass spectrometry structure confirmation

B: When A is O and n is greater than 1

It is necessary to add a long chain synthesis step in the above synthesis step of A (A: when A is O, n=1), that is, after compound 3, first synthesize the long chain and then modify the triphosphate.

Therefore, the steps from compound I to compound III are the same as above. When X is replaced by other groups, only compound 1 needs to be replaced without changing the synthesis process.

From compound III to compound V, different amounts of ethylene oxide are added according to the number of n to control the main compound synthesized, and then the desired compound is purified by Pre-HPLC. The synthesis process of compound VI is the same as the synthesis of compound IV in the synthesis step of A above, the difference is that compound IV is synthesized by taking the synthesized compound III, and compound VI is synthesized by taking the synthesized compound V, and the rest of the process is the same.

This embodiment takes n=5 as an example.

When n=5, except for the amount of compounds I, II, III, and V, the amounts of all added substances are in excess. The difference in the synthesis of different substances is that the amount of the obtained product is different.

Synthesis of Compound V

Weigh 10 mmol of compound III, add it into a clean 100 mL hydrothermal synthesis reactor, and dissolve it with 35 mL of N,N-dimethylformamide; add 60 mmol of ethylene oxide and seal it; after stirring at room temperature for 72 hours, directly spin dry the reaction mixture, and purify the obtained residue by column chromatography, and then separate and purify it by a high-pressure preparative separation and purification system (Pre-HPLC); the obtained eluate is freeze-dried to obtain a white solid.

According to the above method, by changing different X substituents, a series of compounds VI were prepared, and the structures are shown in Table 2;

Table 2 Compounds and mass spectrometry structure confirmation

C: When A is CH ₂ , n＝1

Except for the amount of compounds I, VI, and VII, the amounts of all added substances are in excess. The difference in the synthesis of different substances lies in the different amounts of products obtained.

(1) Synthesis of Compound VII

Weigh 30mmol of compound I and add it to a clean 250mL three-necked flask; add 150mL of N,N-dimethylformamide and replace with nitrogen three times; protect with nitrogen; cool to 0°C in an ice-water bath, weigh 65mmol of sodium hydrogen sulfide (60% in oil) and add it to the reaction system in batches; after adding, heat to 70°C and stir for 2 hours; then cool to 0°C in an ice-water bath; weigh 35mmol of 4-bromobutyl acetate and add it dropwise to the above reaction system; after adding, do not remove the cold bath, naturally heat to room temperature, and stir at room temperature for 48 hours; filter, spin dry the filtrate in a 40°C water bath under high vacuum, and then column chromatography (dichloromethane: methanol = 9:1) to obtain a white solid;

(2) Synthesis of Compound VIII

The compound VII in step (1) was added to a clean 100 mL single-necked flask and dissolved with 50 mL of methanol; the atmosphere was replaced with nitrogen three times and then protected with nitrogen; 1.5 mmol of sodium tert-butoxide was weighed with weighing paper and added to the single-necked flask at one time; the reaction mixture was stirred at room temperature overnight and then directly dried by spin drying. The residue was dispersed in 50 mL of ethyl acetate, acidified with 1N hydrochloric acid, extracted with 50 mL of ethyl acetate four times, and the organic phases were combined and dried over anhydrous sodium sulfate to obtain a white solid.

(3) Synthesis of Compound IX

Weigh 0.25mmol of compound VIII and add it to a clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace the atmosphere with nitrogen three times for nitrogen protection; weigh 0.65mmol of dried pyridine and add it to the above eggplant-shaped bottle at one time; cool the reaction system to 0°C with an ice-water bath; weigh 0.3mmol of phosphorus oxychloride and add it dropwise to the above eggplant-shaped bottle; without removing the cooling bath, stir the above reaction mixture at 0°C for 30 minutes and set it aside as solution A; weigh 0.55mmol of tri-n-butylamine pyrophosphate and add it to another clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace the atmosphere with nitrogen three times for nitrogen protection; The reaction mixture was stirred at room temperature for 2 hours, and the organic solvent was concentrated at 0°C, and the residual aqueous solution was separated and purified by a high-pressure preparative separation and purification system (Pre-HPLC); the obtained eluate was lyophilized to obtain 24.4 mg of a white solid, which was redissolved in 1 mL of cold deionized water and stored at -20°C.

According to the above method, by changing different X substituents, a series of compounds IX were prepared, and the structures are shown in Table 3;

Table 3 Compounds and mass spectrometry structure confirmation

D: When A is CH ₂ and n is greater than 1

Unlike the case when A is O, when A is _CH2 , a long chain needs to be synthesized first and then triphosphate modification is performed.

In this example, n=5. Except for the amount of compounds I, X, and XI, the amounts of all the added substances are in excess. The difference in the synthesis of different substances is that the amount of the obtained product is different.

The specific steps are as follows:

(1) Synthesis of Compound X

Weigh 30 mmol of compound I and add it to a clean 250 mL three-necked flask; add 150 mL of N,N-dimethylformamide and replace with nitrogen three times; protect with nitrogen; cool to 0°C in an ice-water bath, weigh 65 mmol of sodium hydrogen hydride (60% in oil) and add it to the reaction system in batches; after adding, heat to 70°C and stir for 2 hours; then cool to 0°C in an ice-water bath; weigh 25 mmol of 16-bromohexadecyl acetate and add it dropwise to the above reaction system; after adding, do not remove the cold bath at room temperature, naturally heat to room temperature, and stir at room temperature for 48 hours; filter, spin dry the filtrate in a 40°C water bath with high vacuum, and then column chromatography (dichloromethane: methanol = 9:1) to obtain a white solid;

(2) Synthesis of Compound XI

Weigh 2.5 mmol of compound X, add it to a clean 100 mL single-necked flask, and dissolve it with 50 mL of methanol; replace the gas with nitrogen three times and then protect it with nitrogen; weigh 1.2 mmol of sodium tert-butoxide with weighing paper and add it to the single-necked flask at one time; stir the reaction mixture at room temperature overnight and then spin-dry the reaction mixture directly, disperse the residue in 50 mL of ethyl acetate, acidify it with 1N hydrochloric acid, extract it with 50 mL of ethyl acetate four times, combine the organic phases, dry it with anhydrous sodium sulfate and spin-dry it to obtain 0.86 g of a white solid.

(3) Synthesis of Compound XII

Weigh 0.15mmol of compound XI and add it to a clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace the atmosphere with nitrogen three times for nitrogen protection; weigh 0.65mmol of dried pyridine and add it to the above eggplant-shaped bottle at one time; cool the reaction system to 0°C with an ice-water bath; weigh 0.3mmol of phosphorus oxychloride and add it dropwise to the above eggplant-shaped bottle; without removing the cooling bath, stir the above reaction mixture at 0°C for 30 minutes and set it aside as solution A; weigh 0.55mmol of tri-n-butylamine pyrophosphate and add it to another clean 50mL eggplant-shaped bottle; add 2mL of anhydrous acetonitrile and replace the atmosphere with nitrogen three times , nitrogen protection; 1.5mL of dried tri-n-butylamine was measured and added to the above-mentioned eggplant-shaped bottle at one time; the reaction system was cooled to 0°C with an ice-water bath; solution A was added dropwise to the above-mentioned reaction system; without removing the cold bath, the above-mentioned reaction mixture was stirred at 0°C for 30 minutes; 4mL of deionized water was measured and added to the above-mentioned reaction system at one time, and stirred at room temperature for 2 hours; after the organic solvent was concentrated at 0°C, the residual aqueous solution was separated and purified by a high-pressure preparative separation and purification system (Pre-HPLC); the obtained eluate was lyophilized to obtain 11.4mg of white solid, which was redissolved in 1mL of cold deionized water and frozen at -20°C.

According to the above method, by changing different X substituents, a series of compounds XII were prepared, and the structures are shown in Table 4;

Table 4 Compounds and mass spectrometry structure confirmation

Effect Example 1

A: The components of the SNP detection kit are as follows:

(1) Extraction kit components: lysis solution, washing solution I, washing solution II, elution solution, proteinase K, magnetic bead solution.

(2) Multiplex PCR kit components: amplification reaction solution (40 mM Tris-HCl, 800 μM dNTP, 200 nM primer, 8 mM MgCl ₂ ), amplification enzyme solution.

The above extraction kits and multiplex PCR kits were from Chongqing Zhongyuan Huiji Biotechnology Co., Ltd.

(3) Shrimp alkaline phosphatase (SAP enzyme) treatment system: 1 μL 45 mM Tris-HCl, 1 μL 2U/μL SAP enzyme, 1 μL water.

(4) Extension reaction system: 4 μl single base extension reaction solution (45 mM Tris-HCl, 16 μM single base extension primer, 0.6 mM acyNTPs mixed solution), 3 μl 1 U/μL single base extension enzyme.

B: The steps of nucleic acid mass spectrometry detection are as follows:

(1) DNA extraction from samples: DNA was extracted using the components of the Zhongyuan Huiji extraction kit, and the instrument was the Zhongyuan Huiji fully automatic nucleic acid extraction instrument EXM6000.

Multiplex PCR reaction (shown in Table 5):

Table 5 PCR reaction system

Reagent name Volume (μL) Amplification reaction solution 10 Amplification enzyme solution 5 DNA (DNA extracted as above) 5

The reaction volume can be reduced in proportion according to the experimental requirements so that high-throughput detection can be performed through the 384 PCR plate. The prepared multiplex PCR reaction system is amplified on a PCR instrument. The PCR amplification reaction is shown in Table 6:

Table 6 PCR amplification reaction

After PCR amplification, phosphatase digestion was performed at 37°C for 30 min and inactivation was performed at 65°C for 5 min. The reaction volume can be reduced proportionally according to experimental requirements in order to achieve high-throughput detection using a 384 PCR plate.

After digestion is completed, a single base extension reaction is performed. Prepare an extension reaction system, add 7 μl of the extension reaction system to the SAP-digested product, and perform the reaction.

The single base extension reaction settings are shown in Table 7.

Table 7 Single base extension reaction

Resin desalting treatment: Add 20 mg of resin and 30 μl of ddH ₂ O to each reaction well. The volume of resin and ddH ₂ O can be reduced in proportion to the experimental requirements in order to achieve high-throughput detection through the 384 PCR plate. The above resin is purchased from commercial products. Cover the reaction tube (if using a 384 PCR plate, seal it with a sealing film), place it on a rotator and shake it upside down for 5 minutes, then briefly centrifuge.

After desalting is completed, the machine is tested. The mass spectrometer is from Chongqing Zhongyuan Huiji EXS3000 mass spectrometer.

When A is 0, the detection effect is as shown in Example 2-3

Effect Example 2

The structure of the nucleotides in this example is as follows:

The molecular weight is 505.18 and it is named acy sulfo-CTP.

Detection of 281C＞T mutation of SLC26A4, a gene associated with vestibular aqueduct enlargement. SLC26A4 gene mutation can lead to vestibular aqueduct enlargement - simple vestibular aqueduct enlargement or vestibular aqueduct enlargement combined with cochlear malformation. Vestibular aqueduct enlargement is the most common malformation of the inner ear, accounting for 1-8% of hereditary deafness. Clinically, it is mainly manifested as sensorineural deafness with high-frequency hearing loss, and the degree of hearing loss is mostly severe or profound. The disease often occurs in childhood, and before the onset of the disease, there are often predisposing factors such as colds, fevers, and trauma that increase intracranial pressure.

The present invention uses an unmodified nucleotide substrate (reaction 1) and a modified nucleotide substrate (reaction 2) to analyze the 281C>T site of the sample under test.

The unmodified nucleic acid substrate described in this effect example is a mixture of acyATP, acyGTP, acyCTP and acyTTP, and the modified nucleic acid substrate is a mixture of acyATP, acyGTP, acysulfo-CTP and acy TTP.

The results are shown in Table 8 and Figure 1 , which show that the analyzed sample is a patient carrying the mutation gene at the 281C＞T site, which is consistent with the sequencing results.

Table 8 Mutation site analysis

Primers Primer molecular weight (m/z) Extend base Extension product molecular weight Reaction 1/2 Primer1-281C＞T 5873.9 C/T 6069.02/6083.02 Reaction 3 Primer1-281C＞T 5873.9 Sulfonic acid CTP/T 6149.08/6083.02

The sequence of the Primer1-1174A>T is SEQ.ID.NO.1: gtcatttcgggagttagta

The results showed that the modified acy sulfo-CTP was more separated from acyTTP, making it easier to interpret on the spectrum.

Clinical trials

The present invention uses modified nucleic acid substrates (acyATP, acyGTP, acysulfo-CTP and acyTTP mixture) to perform mutation analysis of 20 SNP sites of the deafness gene on the tested samples. The results of SNP typing of deafness-related susceptible genes are shown in Tables 9-11 and Figure 2:

Table 9 Multiplex amplification primers

Table 10 Single base extension primers

serial number sequence Site name SEQ.ID.NO.22 ctccacagtcaagca 1975G>C SEQ.ID.NO.23 aatcctgagaagatgt 1174A>T SEQ.ID.NO.24 caccactgctctttccc 1226G>A SEQ.ID.NO.25 tgttggagtgagatcac 2027T>A SEQ.ID.NO.26 cacgaagatcagctgca 235delC SEQ.ID.NO.27 gcagtagcaattatcgtc IVS7-2A>G SEQ.ID.NO.28 cgtacacaccgcccgtcac 1494C>T SEQ.ID.NO.29 acgtggactgctacattgcc 538C>T SEQ.ID.NO.30 cagcgtggccactagccca 281C>T SEQ.ID.NO.31 cagtgctctcctggacggcc 1229C>T SEQ.ID.NO.32 gatgaacttcctcttcttctc 299_300delAT SEQ.ID.NO.33 ggattagataccccactatgct 1095T>C SEQ.ID.NO.34 tctgtagatagagtatagcatca 2168A>G SEQ.ID.NO.35 tgtctgcaacaccctgcagccag 176_191del16 SEQ.ID.NO.36 tgccagtgccctgactctgctggtt 589G>A SEQ.ID.NO.37 acccctacgcatttatatagaggag 1555A>G SEQ.ID.NO.38 aaaacaaatttctagggataaaata IVS15+5G>A SEQ.ID.NO.39 gggcacgctgcagacgatcctggggg 35delG SEQ.ID.NO.40 ccatgaagtaggtgaagattttcttct 547G>A SEQ.ID.NO.41 aaaggacacattctttttga 2162C>T

Table 11 SNP typing of deafness-related susceptibility genes

The primer set provided by the present invention can be used to type 20 SNP sites of deafness-related susceptibility genes with an accuracy rate of 100%, which is consistent with the results of the first-generation sequencing, the gold standard for gene detection.

Effect Example 3

The structure of the nucleotides in this example is as follows:

Molecular weight 673.44

For convenience, it is named acy-butyl ether-CTP (n=5)

This example explores the detection effect when n>1

This example detects IVS-I-5(G>C), which is a mutation site in β-thalassemia.

Table 12 Mutation site analysis

Primers Primer molecular weight (m/z) Extend base Extension product molecular weight Reaction 1/2 Primer2-IVS-I-5(G>C) 6172.10 G/C 6407.24/6367.22 Reaction 3 Primer2-IVS-I-5(G>C) 6172.10 G/butyl ether-CTP 6407.24/6615.54

The sequence of Primer2-IVS-I-5 (G>C) is SEQ.ID.NO.42: GCAGGTTGAGGCTATCATTA

The results are shown in FIG3 . The modified acy-butylether-CTP (n=5) is very separated from acyGTP, and the results are very easy to interpret.

Effect Example 4: Effect verification when A is _CH2

This example explores the effect of replacing A from O to _CH2 on the detection effect

Because there are many types of nucleotides protected by the present invention, it is impossible to list them all in the examples, so this effect example is explained by studying the following structural formulas.

The molecular weight of the single base extension primer involved in this effect example and the molecular weight of the product after extension with different nucleotide substrates are the same as those in effect example 2.

Table 13 Extension product molecular weight

(1)

The molecular weights were 551.20 and 549.04, respectively, and were named acy I-CTP.

(2)

The molecular weights are 501.22 and 499.25 respectively, and they are named acybenzene-CTP.

(3)

The molecular weights are 523.27 and 521.03 respectively, and they are named acy 1,4-aminopiperidine-CTP

The results are shown in Figure 4, where lines 1, 2, 3, 4, 5, and 6 represent the results of detecting the mutation site 281C＞T using acy I-TTP(A), acy I-TTP(B), acy benzene-TTP(A), acy benzene-TTP(B), acy 1,4-aminopiperidine-CTP(A), and acy 1,4-aminopiperidine-CTP(B) as substrates, respectively.

As can be seen from the figure, in terms of molecular weight, there is not much difference between A and O or _CH2 , so they are closer in the spectrum, but relative to acyCTP, the greater the difference in molecular weight, the closer they can be separated in the spectrum.

Sequence Listing

<110> Zhongyuan Huiji Biotechnology Co., Ltd.

<120> A modified nucleotide, composition and reagent

<130> 2021.10.18

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<213> Synthetic

<400> 7

acgttggatg ctccccagcc tccaccagct tgt 33

<210> 8

<211> 41

<212> DNA

<213> Synthetic

<400> 8

acgttggatg tttgacagtt gttcaagaaa gagagccttt g 41

<210> 9

<211> 34

<212> DNA

<213> Synthetic

<400> 9

acgttggatg cccaggaaga gaactctaag gaag 34

<210> 10

<211> 39

<212> DNA

<213> Synthetic

<400> 10

acgttggatg actatgatag acactgcagc tagagatac 39

<210> 11

<211> 38

<212> DNA

<213> Synthetic

<400> 11

acgttggatg tgatgataag tgagccttaa taagtggg 38

<210> 12

<211> 38

<212> DNA

<213> Synthetic

<400> 12

acgttggatg gcattatttg gttgacaaac aaggaatt 38

<210> 13

<211> 30

<212> DNA

<213> Synthetic

<400> 13

acgttggatggaacaccacactcaccccct 30

<210> 14

<211> 39

<212> DNA

<213> Synthetic

<400> 14

acgttggatg gtaggatcgt tgtcatccag tctcttcct 39

<210> 15

<211> 33

<212> DNA

<213> Synthetic

<400> 15

acgttggatg tgttgccatt cctcgacttg ttc 33

<210> 16

<211> 36

<212> DNA

<213> Synthetic

<400> 16

acgttggatg ggagtgaaga ttcttagatt ttccag 36

<210> 17

<211> 32

<212> DNA

<213> Synthetic

<400> 17

acgttggatg ctattcctga ttggacccca gt 32

<210> 18

<211> 32

<212> DNA

<213> Synthetic

<400> 18

acgttggatg gaacgttccc aaagtgccaa tc 32

<210> 19

<211> 34

<212> DNA

<213> Synthetic

<400> 19

acgttggatg gaaaaccaga accttaccac ccgc 34

<210> 20

<211> 32

<212> DNA

<213> Synthetic

<400> 20

acgttggatg gcaatgcggg ttctttgacg ac 32

<210> 21

<211> 38

<212> DNA

<213> Synthetic

<400> 21

acgttggatg ggaaccttga ccctcttgag atttcact 38

<210> 22

<211> 15

<212> DNA

<213> Synthetic

<400> 22

ctccacagtc aagca 15

<210> 23

<211> 16

<212> DNA

<213> Synthetic

<400> 23

aatcctgaga agatgt 16

<210> 24

<211> 17

<212> DNA

<213> Synthetic

<400> 24

caccactgct ctttccc 17

<210> 25

<211> 17

<212> DNA

<213> Synthetic

<400> 25

tgttggagtg agatcac 17

<210> 26

<211> 17

<212> DNA

<213> Synthetic

<400> 26

cacgaagatc agctgca 17

<210> 27

<211> 18

<212> DNA

<213> Synthetic

<400> 27

gcagtagcaa ttatcgtc 18

<210> 28

<211> 19

<212> DNA

<213> Synthetic

<400> 28

cgtacacacc gcccgtcac 19

<210> 29

<211> 20

<212> DNA

<213> Synthetic

<400> 29

acgtggactg ctacattgcc 20

<210> 30

<211> 19

<212> DNA

<213> Synthetic

<400> 30

cagcgtggcc actagccca 19

<210> 31

<211> 20

<212> DNA

<213> Synthetic

<400> 31

cagtgctctc ctggacggcc 20

<210> 32

<211> 21

<212> DNA

<213> Synthetic

<400> 32

gatgaacttc ctcttcttct c 21

<210> 33

<211> 22

<212> DNA

<213> Synthetic

<400> 33

ggattagata ccccactatg ct 22

<210> 34

<211> 23

<212> DNA

<213> Synthetic

<400> 34

tctgtagata gagtatagca tca 23

<210> 35

<211> 23

<212> DNA

<213> Synthetic

<400> 35

tgtctgcaac accctgcagc cag 23

<210> 36

<211> 25

<212> DNA

<213> Synthetic

<400> 36

tgccagtgcc ctgactctgc tggtt 25

<210> 37

<211> 25

<212> DNA

<213> Synthetic

<400> 37

acccctacgc atttatatag aggag 25

<210> 38

<211> 25

<212> DNA

<213> Synthetic

<400> 38

aaaacaaatt tctagggata aaata 25

<210> 39

<211> 26

<212> DNA

<213> Synthetic

<400> 39

gggcacgctg cagacgatcc tggggg 26

<210> 40

<211> 27

<212> DNA

<213> Synthetic

<400> 40

ccatgaagta ggtgaagatt ttcttct 27

<210> 41

<211> 20

<212> DNA

<213> Synthetic

<400> 41

aaaggacaca ttctttttga 20

<210> 42

<211> 20

<212> DNA

<213> Synthetic

<400> 42

gcaggttgag gctatcatta 20

Claims (5)

1. A modified nucleotide, wherein the modified nucleotide is selected from the group consisting of、、、Or->。

2. A substrate mixture comprising the modified nucleotide of claim 1.

3. The substrate mixture of claim 2, further comprising acyATP, acyGTP and acy UTP, having the following structural formula in order:

、、。

4. a reagent for primer extension comprising the substrate mixture of claim 2 or 3.

5. A kit for mass spectrometry detection of nucleic acids comprising the reagent for primer extension of claim 4.