WO2022161450A1

WO2022161450A1 - Method for measuring relative reaction activity and method for designing oligonucleotide synthesis reaction

Info

Publication number: WO2022161450A1
Application number: PCT/CN2022/074485
Authority: WO
Inventors: 陈锦森; 张有福; 李竑; 王建鹏
Original assignee: 南京金斯瑞生物科技有限公司
Priority date: 2021-01-29
Filing date: 2022-01-28
Publication date: 2022-08-04
Also published as: CN116802319A

Abstract

A method for measuring relative reaction activity and a method for designing an oligonucleotide synthesis reaction. The method for measuring relative reaction activity comprises: (i) selecting any one of N+1 different triplet nucleoside phosphoramidites as an internal standard, the remaining being samples to be measured; (ii) preparing N monomer mixtures containing the internal standard and the samples to be measured; (iii) performing oligonucleotide chain synthesis on the basis of one or more pieces of pre-set oligonucleotide sequence information to obtain synthesized oligonucleotide chains, wherein the one or more oligonucleotide sequences comprise at least N activity test points corresponding to the N monomer mixtures; iv) sequencing the synthesized oligonucleotide chains; and (v) determining the relative reaction activity ratio of the internal standard to each sample to be measured on the basis of the sequencing results.

Description

Relative reactivity determination method and design method of oligonucleotide synthesis reaction

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No. 202110125872.0 filed on January 29, 2021, the entire contents of which are incorporated herein by reference.

technical field

The present specification relates to the field of chemical synthesis of oligonucleotides, in particular to a method for measuring the relative reactivity of triple nucleoside phosphoramidites and a method for designing oligonucleotide synthesis reactions.

Background technique

In recent years, protein research at the molecular and submolecular levels has been greatly developed, in which combinatorial gene synthesis plays an important role in limiting structural variation. Oligonucleotide-directed mutagenesis is the most common method for preparing polypeptide or protein variants of interest. Oligonucleotides of mixed composition are increasingly used to generate variant libraries for studying the function of biomolecules and to find peptides and proteins with improved properties. Among the many library preparation methods, degenerate oligonucleotides or oligonucleotide libraries, but this method cannot avoid the incorporation of unwanted amino acids and stop codons, nor can it construct the desired codon subsets at predetermined positions. Using a preformed mixture of 20 triplet nucleoside phosphoramidites representing canonical amino acid codons, any number of codon variations can be made at any codon position in a given gene, resulting in a high-quality mutation library. Therefore, the demand for this triple nucleoside phosphoramidite is growing rapidly.

When synthesizing oligonucleotide chains by solid-phase synthesis, different triple nucleoside phosphoramidites show different coupling efficiencies. In order to make the codon distribution in the library uniform or adjust the mutation rate, the triple nucleoside phosphoramidites must be considered. Different reactivity of phosphoramides to prepare trinucleotide mixtures. Therefore, it is necessary to propose a simple, rapid and low-cost method for determining the relative reactivity of triple nucleoside phosphoramidites.

SUMMARY OF THE INVENTION

One of the embodiments of this specification provides a method for determining the relative reactivity of trinucleoside phosphoramidites, the method comprising:

ⅰ) Select any one of N+1 different triple nucleoside phosphoramidites as the internal standard, and the remaining N triple nucleoside phosphoramidites as the test samples, where N is an integer and N≥1;

ii) Prepare N monomer mixtures, wherein each of the monomer mixtures includes the internal standard product and one of the test products, and the internal standard product and the test product in each of the monomer mixtures The molar ratios of the products are the same, and the test products in each of the monomer mixtures are different from each other;

iii) synthesizing an oligonucleotide chain based on the preset sequence information of one or more oligonucleotides to obtain a synthetic oligonucleotide chain, wherein, by setting the incorporation sites of the N monomer mixtures, causing the one or more oligonucleotide sequences to include at least N active test points, each of the monomer mixtures having a corresponding at least one active test point;

iv) sequencing the synthesized oligonucleotide strand, and

ⅴ) Determine the relative reactivity ratio of the internal standard product to each test product based on the sequencing results.

In some embodiments, the oligonucleotide sequence has a test region and a non-test region, the at least N active test spots are distributed in the test region of the one or more oligonucleotide sequences, and any two A spacer sequence comprising at least one nucleotide is independently present or absent between adjacent active test points.

In some embodiments, the one or more oligonucleotide sequences include N active test sites randomly incorporated by each of the N monomer mixtures The one or more oligonucleotide sequences are formed by reacting the test regions; or, the one or more oligonucleotide sequences include N active test points, and the N active test points are composed of the N active test points. Each monomer mixture in the monomer mixture is formed by reacting with a nucleotide coupling of the same base.

In some embodiments, the one or more oligonucleotide sequences comprise 2N active test sites, the 2N active test sites being separated from each of the N monomer mixtures by 2 It is formed by the reaction of nucleotide coupling of different bases.

In some embodiments, the one or more oligonucleotide sequences comprise 3N active test sites, the 3N active test sites being formed by each of the N monomer mixtures with 3 It is formed by the reaction of nucleotide coupling of different bases.

In some embodiments, the one or more oligonucleotide sequences comprise 4N active test points, the 4N active test points are formed by each monomer mixture of the N monomer mixtures and 4 It is formed by the reaction of nucleotide coupling of different bases.

In some embodiments, any triple nucleoside phosphoramidite in the N+1 triple nucleoside phosphoramidites corresponds to a codon encoding an amino acid, and in the N+1 triple nucleoside phosphoramidites The codons corresponding to any two triplets of nucleoside phosphoramidites encode different amino acids.

In some embodiments, the N is any positive integer within 18.

In some embodiments, the N is 19.

In some embodiments, the selected internal standard is AAC phosphoramidite.

In some embodiments, the determining the relative reactivity ratio between the internal standard product and each test product based on the sequencing result includes: counting the internal standard product readings and the test product corresponding to the at least N activity test points in the sequencing result respectively Readings, the ratio of the readings of the internal standard substance to the readings of the test substance at each activity test point is the relative reactivity ratio of the internal standard substance to the test substance corresponding to the activity test point.

In some embodiments, the method further comprises: vi) determining the relative reactivity coefficient of each test article based on the relative reactivity ratio.

In some embodiments, the molar ratio of the internal standard to the test substance in each monomer mixture is 1:1.

In some embodiments, the sequencing the synthesized oligonucleotide strand comprises: sequencing the synthesized oligonucleotide strand using next generation sequencing technology.

In some embodiments, the performing oligonucleotide strand synthesis based on the preset one or more oligonucleotide sequence information includes: performing oligonucleotide strand synthesis using a solid-phase synthesis method in a DNA synthesizer.

In some embodiments, the use of the relative reactivity of the triple nucleoside phosphoramidite determined by the above method in the construction of a gene library. In a specific embodiment, the use of the relative reactivity of the triple nucleoside phosphoramidite determined by the above method in the construction of a gene mutation library. Using the relative reactivity of the triple nucleoside phosphoramidites determined by the above method to synthesize nucleic acid sequences containing specific site mutations, the mutation sites can be randomly distributed 20 triple nucleoside phosphoramidites representing canonical amino acid codons in the nucleic acid sequence one or more of. Specifically, the relative reactivity coefficient of the triple nucleoside phosphoramidite is used to determine the relative reactivity coefficient of the triple nucleoside phosphoramidite, and the ratio of the added amount of the triple nucleoside phosphoramidite corresponding to each site and/or the mutation site is further confirmed, according to In this ratio, a variety of triple nucleoside phosphoramidites are used as synthetic raw materials to synthesize to a predetermined position in the nucleic acid sequence, and a high-quality DNA library conforming to the predetermined nucleic acid sequence can be obtained, such as a primer library, a mutation library and a gene element combination library.

One of the embodiments of this specification provides a method for designing an oligonucleotide synthesis reaction, the method comprising:

ii) determining the relative reactivity ratio of the internal standard to each test article; and

iii) Determine the ratio of triple nucleoside phosphoramidites used in the reaction of synthesizing oligonucleotides according to the relative reactivity ratio of the internal standard substance and each test substance.

In some embodiments, the determining the relative reactivity ratio of the internal standard to each test product comprises:

Prepare N monomer mixtures, wherein each of the monomer mixtures includes the internal standard and one of the samples to be tested, and the internal standard and the sample to be tested in each of the monomer mixtures are different from each other. The molar ratios are the same, and the samples to be tested in each of the monomer mixtures are different from each other;

Perform oligonucleotide chain synthesis based on preset one or more oligonucleotide sequence information to obtain a synthesized oligonucleotide chain, wherein by setting the incorporation sites of the N monomer mixtures, all The one or more oligonucleotide sequences include at least N activity test points, and each of the monomer mixtures has a corresponding at least one activity test point;

sequencing the synthesized oligonucleotide chain, and

Based on the sequencing results, the relative reactivity ratio of the internal standard product to each test product was determined.

In some embodiments, AAC phosphoramidites among the 20 triple nucleoside phosphoramidites representing canonical amino acid codons are selected as internal standards, and the remaining AAA phosphoramidites, ACT phosphoramidites, ATC phosphoramidites, ATG phosphoramidite, CAG phosphoramidite, CAT phosphoramidite, CCG phosphoramidite, CGT phosphoramidite, CTG phosphoramidite, GAA phosphoramidite, GAC phosphoramidite, GCT phosphoramidite, GGT phosphoramidite, GTT phosphoramidites, TAC phosphoramidites, TCT phosphoramidites, TGC phosphoramidites, TGG phosphoramidites and TTC phosphoramidites are the samples to be tested.

One of the embodiments of the present specification provides a method for designing an oligonucleotide synthesis reaction, the method comprising: selecting an AAC phosphoramidite among 20 triplet nucleoside phosphoramidites representing canonical amino acid codons as an internal standard , and the remaining 19 are the samples to be tested. Determine the relative reactivity coefficient of each test product based on the relative reactivity ratio of the internal standard product and each test product as shown in the table, and determine according to the relative reactivity coefficient. Proportion of triple nucleoside phosphoramidites used in oligonucleotide synthesis reactions.

One of the examples in this specification provides the relative reactivity coefficients of the triple nucleoside phosphoramidites. The AAC phosphoramidite among the 20 triple nucleoside phosphoramidites representing canonical amino acid codons is selected as the internal standard, and the remaining 19 are selected as the internal standard. For the test article, determine the relative reactivity coefficient of each test article calculated based on the relative reactivity ratio of the internal standard to each test article as shown in the table below.

One of the embodiments of the present specification also provides the application of the above-mentioned relative reactivity coefficient of the triple nucleoside phosphoramidite in constructing a gene library. The ratio of the added amount of the corresponding tripartite nucleoside phosphoramidites at each site and/or the mutation site is determined by using the tripartite nucleoside phosphoramidite relative to its reactivity coefficient, and a variety of tripartite nucleoside phosphoramidites are used as synthetic raw materials according to the ratio. After synthesizing to a predetermined position in the nucleic acid sequence, a high-quality DNA library conforming to the predetermined nucleic acid sequence can be obtained, such as a primer library, a mutation library and a gene element combinatorial library.

Description of drawings

The present specification will be further described by way of example embodiments, which will be described in detail with reference to the accompanying drawings. These examples are not limiting, and in these examples, the same numbers refer to the same structures, wherein:

Fig. 1 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq1 shown in some embodiments of the present specification;

Fig. 2 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq2 shown in some embodiments of the present specification;

Fig. 3 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq3 shown in some embodiments of the present specification;

Fig. 4 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq4 shown in some embodiments of the present specification;

Fig. 5 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq5 shown in some embodiments of the present specification;

Fig. 6 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq6 shown in some embodiments of the present specification;

Fig. 7 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq7 shown in some embodiments of the present specification;

Fig. 8 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq8 shown in some embodiments of the present specification;

Fig. 9 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq9 shown in some embodiments of the present specification;

Figure 10 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq10 shown in some embodiments of the present specification;

Fig. 11 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq11 shown in some embodiments of the present specification;

Fig. 12 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq12 shown in some embodiments of the present specification;

Fig. 13 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq13 shown in some embodiments of the present specification;

Fig. 14 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq14 shown in some embodiments of the present specification;

Fig. 15 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq15 shown in some embodiments of the present specification;

Figure 16 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq16 shown in some embodiments of the present specification;

Figure 17 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq17 shown in some embodiments of the present specification;

Figure 18 is an HPLC analysis map of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq18 shown in some embodiments of the present specification;

Fig. 19 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq19 shown in some embodiments of the present specification;

Figure 20 is an HPLC analysis map of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq20 shown in some embodiments of the present specification;

Figure 21 is the HPLC analysis pattern of the pure oligonucleotide obtained by separation and purification of the crude oligonucleotide synthesized based on the oligonucleotide sequence Seq21 according to some embodiments of the present specification.

Detailed ways

In order to more clearly illustrate the technical solutions of the embodiments of the present application, one or some embodiments in the present specification will be briefly introduced below. Obviously, the following descriptions are only some examples or embodiments of the present application. For those of ordinary skill in the art, the present application can also be applied to other similar situations according to these embodiments without creative efforts. .

As shown in this application and in the claims, unless the context clearly dictates otherwise, the words "a", "an", "an" and/or "the" are not intended to be specific in the singular and may include the plural. Generally speaking, the terms "comprising" and "comprising" only imply that the clearly identified steps and elements are included, and these steps and elements do not constitute an exclusive list, and the method or apparatus may also include other steps or elements.

The following are definitions of some terms in this application.

The term "nucleoside phosphoramidites" refers to phosphoramidites obtained by chemically modifying nucleotides, and is the raw material for solid-phase synthesis of oligonucleotides, which can include mononucleoside phosphoramidites, dinucleoside phosphoramidites and triple nuclei. glycoside phosphoramidites, etc.

The term "canonical amino acid codons" refers to 20 codons that encode amino acids out of 64 codons selected for ease of industrial application. Among the selected 20 canonical amino acid codons, the amino acids encoded by each canonical amino acid codon are different from each other. The 20 canonical amino acid codons are AAA, AAC, ACU, AUC, AUG, CAG, CAU, CCG, CGU, CUG, GAA, GAC, GCU, GGU, GUU, UAC, UCU, UGC, UGG, and UUC, respectively.

The term "polymerase chain reaction (PCR)" refers to a nucleic acid synthesis technique that utilizes the principle of DNA double-strand replication to replicate specific DNA fragments in vitro. Through this technology, a large number of target genes can be amplified in a short time.

The term "gene mutation library" refers to the nucleic acid coding sequence of one or more proteins, and one or more amino acid sites are mutated into other 19/20 amino acids through mutation, and the corresponding nucleic acid sequences formed library.

The term "combinatorial library of genetic elements" refers to the accurate and efficient assembly and cloning of various predefined genetic elements (DNA sequences) into a vector of choice to generate a library of cloning constructs assembled in a specific arrangement. These predefined genetic elements (DNA sequences) may be promoters, enhancers/repressors, specific binding sites, localization signals, genes, terminators, and the like.

In the construction of DNA libraries, the use of triple nucleoside phosphoramidites to obtain high-quality libraries has been widely used. Nucleotide sequences with any number of codon variations at any codon position can be synthesized by using a pre-prepared mixture of 20 triplet nucleoside phosphoramidites representing canonical amino acid codons. The basic steps for the synthesis of oligonucleotide chains by triple nucleoside phosphoramidites include: 1) Deprotection group: use trichloroacetic acid to remove the protective group dimethoxytrityl of the nucleotides attached to the solid support 2) Activation: Mix the trinucleoside phosphoramidite with the tetrazolium activator and enter the solid phase carrier to form the phosphoramidite tetrazolium active intermediate body (its 3'-end has been activated, but the 5'-end is still protected by the protecting group dimethoxytrityl), this intermediate will interact with the deprotected nucleotides on the solid support Condensation reaction occurs; 3) Connection: when the phosphoramidite tetrazole active intermediate encounters the nucleotide whose deprotected group is on the solid phase carrier, the phosphorous acid group at the 3' end of the phosphoramidite tetrazole active intermediate will interact with the solid phase carrier. The 5'-hydroxyl nucleotide of the deprotected group on the phase carrier undergoes an affinity reaction, condenses and removes the tetrazole, and the synthesized oligonucleotide chain is extended forward by three nucleotides at this time; 4) Blocking: After the condensation reaction, in order to prevent the unreacted deprotected nucleotide 5'-hydroxyl group attached to the solid support from being extended in the subsequent cyclic reaction, acetylation is often used to block the unreacted 5'-hydroxyl group. Hydroxyl end; 5) Oxidation: During the condensation reaction, the triple nucleotide monomer is connected to the oligonucleotide attached to the solid support through a phosphite bond, and the tetrahydrofuran solution of iodine is commonly used to convert the phosphite triester into phosphoric acid Triesters, resulting in stable oligonucleotide chains. The above steps allow a triple nucleotide to be linked to the 5' end of the nucleotides of the solid support, and the above steps are repeated to obtain the target oligonucleotide chain. After the synthesis, cleavage of the oligonucleotide chain and an abasic protection group operation can be performed, and then the synthesized oligonucleotide chain can be obtained through separation and purification. It is clear that in the process of oligonucleotide chain synthesis, the coupling efficiencies of different tripartite nucleoside phosphoramidites and deprotected nucleotide 5'-hydroxyl groups are different. In the process of constructing a library by using triple nucleoside phosphoramidites, the reactivity of different triple nucleoside phosphoramidites needs to be determined to improve the quality of the library.

This specification provides a method for determining the relative reactivity of triple nucleoside phosphoramidites. This method can select one of several triple phosphoramidites to be used as the internal standard, and the triple phosphoramidite other than the internal standard As the test product, the internal standard is the reference for the test product to compare the reactivity. The sample to be tested is separately mixed with the internal standard substance in the same proportion to form a monomer mixture, and the monomer mixture is used to synthesize an oligonucleotide chain including a predetermined active test point. The content determination and comparison of each activity test point carry out the relative quantitative characterization of the reactivity of the test product. The relative quantitative characterization of the reactivity of the test product is carried out by measuring and comparing the contents of the internal standard product and the test product at each activity test point in the oligonucleotide chain. Further, the activity test point can be set to reflect the coupling efficiency of the internal standard and the test product with nucleotides of different bases under the same synthesis conditions, that is, to reflect the difference between the 3' ends of the internal standard and the test product. The reactivity of nucleotides (adenine deoxynucleotides; guanine deoxynucleotides; cytosine deoxynucleotides; thymidine deoxynucleotides) in the 5'-terminal hydroxyl affinity reaction makes it easier to characterize relative reactivity to be accurate. Applicable aspects of the above-described method for determining the relative reactivity of trinucleoside phosphoramidite include, but are not limited to, construction of DNA libraries, polypeptide libraries, antibody libraries and protein libraries.

It should be understood that the application scenarios of the method for determining the relative reactivity of the triple nucleoside phosphoramidite in the present application are only some examples or embodiments of the present application. On the premise, the present application can also be applied to other similar scenarios according to these drawings.

The present invention provides a method for measuring the relative reactivity of triple nucleoside phosphoramidites, which at least comprises the following steps:

Step ⅰ, select the product to be tested and the internal standard product.

In some embodiments, selecting the sample to be tested and the internal standard includes: selecting any triple nucleoside phosphoramidite as the internal standard among the N+1 triple nucleoside phosphoramidites to be determined, and the remaining N The triple nucleoside phosphoramidite was used as the test substance, and N≥1. In some embodiments, the N+1 triplet nucleoside phosphoramidites can be at least 2 triplet nucleoside phosphoramidites of the 64 triplet nucleoside phosphoramidites representing codons. Specifically, the internal standard can be a reference for the relative reactivity of the analyte, and can be used to reflect that the relative reactivity of the analyte is higher than, equal to, or lower than the relative reactivity of the internal standard. Any one of the measured triple nucleoside phosphoramidites, and as a reference, the relative reactivity ratio and relative reactivity coefficient of the internal standard can be marked as 1.

In some embodiments, any triple nucleoside phosphoramidite in the N+1 triple nucleoside phosphoramidites corresponds to a codon encoding an amino acid, and in the N+1 triple nucleoside phosphoramidites The codons corresponding to any two triplet of nucleoside phosphoramidites encode different amino acids. In some embodiments, further, the N+1 triplet nucleoside phosphoramidites may be at least 2 triplet nucleoside phosphoramidites among the 20 triplet nucleoside phosphoramidites representing canonical amino acid codons. Specifically, the sequence composition and amino acid correspondence of the 20 triplet nucleoside phosphoramidites representing canonical amino acid codons are shown in Table 1.

Table 1. Enantiomeric relationship between triple nucleoside phosphoramidites and amino acids

In some embodiments, the number N of DUTs can be any positive integer within 18. For example, the number N of the samples to be tested may be 18, and the N+1 triplet nucleoside phosphoramidites may be any 19 of the 20 triplet nucleoside phosphoramidites representing canonical amino acid codons; wherein, the internal standard It can be any one of the 19 triple nucleoside phosphoramidites, and the other 18 triple nucleoside phosphoramidites are the samples to be tested. For example, the number N of the test substance can be 2, and the N+1 triplet nucleoside phosphoramidites can be any 3 of the 20 triplet nucleoside phosphoramidites representing canonical amino acid codons; wherein, the internal standard It can be any one of the three triple nucleoside phosphoramidites, and the other two triple nucleoside phosphoramidites are the samples to be tested. For example, the number N of the test substance can be 1, and N+1 triplet nucleoside phosphoramidites can be any 2 of the 20 triplet nucleoside phosphoramidites representing canonical amino acid codons; wherein, the internal standard It can be any one of the two triple nucleoside phosphoramidites, and the remaining one triple nucleoside phosphoramidite is the sample to be tested.

In some embodiments, the number N of DUTs may be 19. Specifically, N can be 19, and N+1 triple nucleoside phosphoramidites can be 20 triple nucleoside phosphoramidites representing canonical amino acid codons; wherein, the internal standard can be AAA phosphoramidite, or AAC Phosphoramidite, or ACT phosphoramidite, or ATC phosphoramidite, or ATG phosphoramidite, or CAG phosphoramidite, or CAT phosphoramidite, or CCG phosphoramidite, or CGT phosphoramidite, or CTG phosphoramidite amide, or GAA phosphoramidite, or GAC phosphoramidite, or GCT phosphoramidite, or GGT phosphoramidite, or GTT phosphoramidite, or TAC phosphoramidite, or TCT phosphoramidite, or TGC phosphoramidite, Or TGG phosphoramidite, or TTC phosphoramidite, the test substance can be 19 triple nucleoside phosphoramidites except the internal standard.

In some embodiments, further, the selected internal standard is AAC phosphoramidite. For example, the selected internal standard is AAC phosphoramidite, the number N of the analyte can be 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19, and the N different triple nucleoside phosphoramidites as the test substance can be selected from the 20 triple nucleoside phosphoramidites representing canonical amino acid codons except AAC phosphoramidites Any N triplet nucleoside phosphoramidites other than .

In step ii, a monomer mixture is prepared.

The monomer mixture is the mixture of the internal standard and a single test substance, and the monomer mixture can be used as a reactant for synthesizing oligonucleotide chains.

In some embodiments, preparing the monomer mixture includes preparing N monomer mixtures, each monomer mixture is a mixture of an internal standard and a test product, and the test products in each monomer mixture are different from each other. In some embodiments, the molar ratio of the internal standard to the test substance in each monomer mixture is the same. For example, the molar ratio of internal standard to test substance in each monomer mixture can be 1:4, or 3:7, or 2:3, or 1:1, or 3:2, or 7:3, or 4:1. In some embodiments, for the convenience of statistical analysis, the molar ratio of the internal standard substance to the test substance in each mixed reaction is 1:1.

In step iii, the oligonucleotide chain is synthesized using the monomer mixture.

The oligonucleotide chain is synthesized by using the monomer mixture as the raw material. In the synthesized oligonucleotide chain, one monomer mixture incorporation site of part of the oligonucleotide chain can be incorporated into the internal standard, and the rest of the oligonucleotide chain can be incorporated into the internal standard. The corresponding monomer mixture incorporation sites of the analytes can be incorporated. The internal standard is used as a reference to reflect the relative coupling efficiency of the test product under the same synthesis conditions.

In some embodiments, using the monomer mixture to synthesize an oligonucleotide chain includes: performing an oligonucleotide chain synthesis based on preset one or more oligonucleotide sequence information to obtain a synthesized oligonucleotide chain, wherein , by setting the incorporation sites of the N monomer mixtures, so that the one or more oligonucleotide sequences include at least N activity test points, and each of the monomer mixtures has a corresponding at least one activity test point. Specifically, one or more oligonucleotide sequences can be provided with N or 2N or 3N or 4N incorporation sites, and each monomer mixture can correspond to 1 or 2 or 3 or 4 incorporation sites, respectively, so that all The one or more oligonucleotide sequences can respectively include N or 2N or 3N or 4N active test points respectively; wherein, the active test points can be a specific couple of nucleotides to which the monomer mixture is coupled to a specific base. Coupling activity test point, the activity test point may also be a non-specific coupling activity test point that does not examine the coupling of the monomer mixture to a nucleotide of a specific base.

In some embodiments, the oligonucleotide sequence can have a test region and a non-test region. Specifically, if an oligonucleotide chain is synthesized based on a preset oligonucleotide sequence information, the at least N active test points are distributed in a test area of an oligonucleotide sequence; if based on a preset at least 2 oligonucleotide sequence information to synthesize an oligonucleotide chain, the test area of each oligonucleotide sequence is distributed with some active test points in the at least N active test points; when the number of oligonucleotide sequences When greater than 1, the non-test regions of each oligonucleotide sequence are the same, that is, the nucleotide sequences of the non-test regions of each oligonucleotide sequence are the same; in some embodiments, the same oligonucleotide In the sequence, the test region can be located between two non-test regions, and the two non-test regions are respectively provided with primer binding sites for PCR amplification. Specifically, when the synthesized oligonucleotide chain is subjected to PCR amplification and sequencing, the forward primer used for PCR amplification can bind to the primer binding site of one of the two test regions, and the reverse primer can bind to the primer binding site of one of the two test regions. Binds to the primer binding site of another test region.

In some embodiments, further, the at least N active test points are distributed in the test region of the one or more oligonucleotide sequences; any two adjacent active test points exist independently or not There is a spacer sequence comprising at least one nucleotide. Specifically, multiple active test points within the test area of the same oligonucleotide sequence can be arranged in any order; for the active test points in the same oligonucleotide sequence, different adjacent active test points can be arranged in any order. The spacer sequence comprising at least one nucleotide is independently present or absent; for active test spots in different oligonucleotide sequences, the presence or absence of at least one active test spot between different adjacent active test spots may be independently present or absent. Nucleotide spacer sequence.

In some embodiments, the oligonucleotide sequence comprises a test region and a non-test region, and the one or more oligonucleotide sequences comprise N active test sites, the N active test sites consisting of the N Each of the monomer mixtures is formed by reacting randomly incorporating the one or more oligonucleotide sequences into the test region. Specifically, the N activity test points belong to non-specific coupling activity test points. In some embodiments, further, the one or more oligonucleotide sequences include N activity test points, and the N activity test points are randomly selected from each of the N monomer mixtures. formed by reacting a test region that incorporates the one or more oligonucleotide sequences; the N monomer mixtures are numbered from 1 to N, each oligonucleotide in the one or more oligonucleotide sequences Both nucleotide sequences can have the following structures:

5'-L ₁ -M[X _i ]-L ₂ -3'.

Wherein, L1 and L2 are each independently a sequence comprising at least 15 nucleotides, L1 and L2 represent the non-test region of the oligonucleotide sequence, and the sequences between L1 and L2 represent the test region of the oligonucleotide sequence;

M is the number of sequence units containing Xi in the test region, M is an integer and 1≤M≤N, and there is or does not independently exist a spacer sequence comprising at least one nucleotide between adjacent sequence units;

Xi is the monomer mixture corresponding to the incorporation site, i represents the number of the monomer mixture, i=1, 2, ..., N.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset An oligonucleotide sequence information of an oligonucleotide chain is synthesized, and the oligonucleotide sequence is provided with 4 incorporation sites of the monomer mixture, so that the oligonucleotide sequence includes 4 active test points, 4 The active test point belongs to the non-specific coupling active test point, and the oligonucleotide sequence can be:

5'-L ₁ -X ₁ ATX ₂ X ₃ CX ₄ -L _2-3 '.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The 2 oligonucleotide sequence information of the synthetic oligonucleotide chain, the 2 oligonucleotide sequences are provided with the incorporation sites of 4 monomer mixtures, so that the 2 oligonucleotide sequences include 4 active test sites , 4 activity test points belong to non-specific coupling activity test points, and the 2 oligonucleotide sequences can be respectively:

₁ ) 5'-L1 _- _X1X3 _- L2-3';

₂ ) 5'-L1 _- _X2X4 _- L2-3'.

In some embodiments, the one or more oligonucleotide sequences comprise N active test sites, the N active test sites being identical to one each from each of the N monomer mixtures Formed by the reaction of nucleotide coupling of bases. Specifically, the N activity test points belong to a specific coupling activity test point. For example, N active test sites are formed by each of N monomer mixtures coupled to adenine deoxynucleotides, or N active test sites are formed by each monomer in N monomer mixtures The mixture is formed by coupling guanine deoxynucleotides, or N active test sites are formed by coupling each monomer mixture of N monomer mixtures with cytosine deoxynucleotides, or N active test sites Formed by coupling each of the N monomer mixtures with thymidine. In some embodiments, further, the one or more oligonucleotide sequences include N activity test points, and the N activity test points are separated from each monomer mixture in the N monomer mixtures. Formed by reacting with one conjugated test nucleotide; the conjugated test nucleotide can be any one of 4 different base nucleotides, numbered from 1 to N for the N monomer mixture, the one or Each of the plurality of oligonucleotide sequences can have the following structure:

5'-L ₁ -M[X _i Y]-L ₂ -3'.

M is the number of sequence units containing Xi and Y in the test region, M is an integer and 1≤M≤N, the presence or absence of a spacer sequence comprising at least one nucleotide between adjacent sequence units;

Xi is the monomer mixture corresponding to the incorporation site, i represents the number of the monomer mixture, i=1, 2, ..., N;

Y is the coupling test nucleotide, and Y is any one of the nucleotides of 4 different bases.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset An oligonucleotide sequence information of an oligonucleotide chain is synthesized, and the oligonucleotide sequence is provided with 4 incorporation sites of the monomer mixture, so that the oligonucleotide sequence includes 4 active test points, 4 The active test point belongs to a specific coupling active test point, and each active test point is formed by coupling 4 monomer mixtures with thymidine respectively; the oligonucleotide sequence can be:

5'-L ₁ -X ₁ TX ₂ TX ₃ TX ₄ TL _2-3 '.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The 2 oligonucleotide sequence information of the synthetic oligonucleotide chain, the 2 oligonucleotide sequences are provided with the incorporation sites of 4 monomer mixtures, so that the 2 oligonucleotide sequences include 4 active test sites , 4 active test points belong to specific coupling activity test points, each active test point is formed by the coupling of 4 monomer mixtures with guanine deoxynucleotides respectively; the 2 oligonucleotide sequences can be respectively:

1) 5'-L ₁ -X ₁ GX ₃ GTAX ₄ GL _2-3 ';

₂ ) 5'-L1 _- _X2GL2-3 '.

In some embodiments, the one or more oligonucleotide sequences comprise 2N active test sites, the 2N active test sites being separated from each of the N monomer mixtures by 2 It is formed by the reaction of nucleotide coupling of different bases. Specifically, the 2N active test points belong to a specific coupling active test point, and the nucleotides with two different bases can be selected from any two of the nucleotides with four different bases. In some embodiments, further, the one or more oligonucleotide sequences include 2N active test points, and the 2N active test points are respectively different from 2 by each of the N monomer mixtures The coupling test of bases is formed by the reaction of nucleotide coupling; the coupling test nucleotides of 2 different bases can be any 2 of the nucleotides of 4 different bases, and the coupling test of 2 Nucleotides are numbered from 1 to 2, and N monomer mixtures are numbered from 1 to N; each of the one or more oligonucleotide sequences may have the following structure:

5'-L ₁ -M[X _i Y _j ]-L ₂ -3'.

M is the number of sequence units containing Xi and Yj in the test region, M is an integer and 1≤M≤2N, and there is or does not exist a spacer sequence comprising at least one nucleotide between adjacent sequence units;

Xi is the monomer mixture corresponding to the incorporation site, i represents the number of the monomer mixture, and i=1, 2, , N;

Yj is the conjugated test nucleotide, j represents the number of the conjugated test nucleotide, and j=1,2.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The oligonucleotide chain is synthesized with the information of an oligonucleotide sequence, the oligonucleotide sequence is provided with 8 incorporation sites, and each monomer mixture has corresponding 2 incorporation sites, so that the oligonucleotide The acid sequence includes 8 active test points, 8 active test points belong to specific coupling activity test points, and 8 active test points are respectively coupled with adenine deoxynucleotide and thymidine deoxynucleotide by 4 monomer mixtures. formed; the oligonucleotide sequence may be:

5'-L ₁ -X ₁ TCCX ₁ AX ₂ TAAX ₂ AX ₃ TGGX ₃ AX ₄ TTTX ₄ AL _2-3 '.

For example, one of the 5 triplet nucleoside phosphoramidites is selected as the internal standard, and the remaining 4 are the samples to be tested, and 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ are prepared, which can be based on the preset 4 oligonucleotide sequence information to synthesize oligonucleotide chain, 4 oligonucleotide sequences are set with 8 incorporation sites, and each monomer mixture has corresponding 2 incorporation sites, so that 4 oligonucleotides The nucleotide sequence includes 8 active test points, 8 active test points belong to specific coupling activity test points, and 8 active test points are composed of 4 monomer mixtures with guanine deoxynucleotide and cytosine deoxynucleotide respectively. formed by coupling; the 4 oligonucleotide sequences can be respectively:

1) 5'-L ₁ -X ₁ GX ₂ GL _2-3 ';

2) 5'-L ₁ -X ₃ GX ₄ GL _2-3 ';

₃ ) 5' _- L1 _- _X1CX2CL2-3 ';

₄ ) 5' _- L1 _- _X3CX4CL2-3 '.

In some embodiments, the one or more oligonucleotide sequences comprise 3N active test sites, the 3N active test sites being formed by each of the N monomer mixtures with 3 It is formed by the reaction of nucleotide coupling of different bases. Specifically, the 3N active test points belong to a specific coupling active test point, and the nucleotides with 3 different bases can be selected from any 3 of the nucleotides with 4 different bases. In some embodiments, further, the one or more oligonucleotide sequences include 3N active test points, and the 3N active test points are respectively different from 3 by each of the N monomer mixtures The coupling test of bases is formed by the reaction of nucleotide coupling; the coupling test nucleotides of 3 different bases can be any 3 of the nucleotides of 4 different bases; the coupling test of 3 different bases Nucleotides are numbered from 1 to 3, and N monomer mixtures are numbered from 1 to N, each of the one or more oligonucleotide sequences may have the following structure:

5'-L ₁ -M[X _i Y _j ]-L ₂ -3'.

M is the number of sequence units containing Xi and Yj in the test region, M is an integer and 1≤M≤3N, and there is or does not exist a spacer sequence comprising at least one nucleotide between adjacent sequence units;

Yj is the conjugated test nucleotide, j represents the number of the conjugated test nucleotide, and j=1, 2, 3.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The oligonucleotide chain is synthesized with the sequence information of an oligonucleotide, the oligonucleotide sequence is provided with 12 incorporation sites, and each monomer mixture has corresponding 3 incorporation sites, so that the oligonucleotide The acid sequence includes 12 active test points, 12 active test points belong to specific coupling active test points, and 12 active test points are composed of 4 monomer mixtures with adenine deoxynucleotide, guanine deoxynucleotide and thymus, respectively. formed by coupling of pyrimidine deoxynucleotides; the oligonucleotide sequence can be:

5'-L ₁ -X ₁ TX ₂ TX ₃ TX ₄ TX ₁ GX ₂ GX ₃ GX ₄ GX ₁ AX ₂ AX ₃ AX ₄ AL ₂ -3'.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The 3 oligonucleotide sequence information of the synthetic oligonucleotide chain, the 3 oligonucleotide sequences are set with 12 incorporation sites, and each monomer mixture has corresponding 3 incorporation sites, so that 3 The oligonucleotide sequence includes 12 active test points, 12 active test points belong to specific coupling active test points, and 12 active test points are composed of 4 monomer mixtures with guanine deoxynucleotide, cytosine deoxynucleoside respectively. formed by the coupling of acid and thymidine deoxynucleotides; the 3 oligonucleotide sequences can be respectively:

1) 5'-L ₁ -X ₁ CX ₂ CTX ₃ CX ₄ CA-L _2-3 ';

2) 5'-L ₁ -X ₁ GTTX ₂ GCCX ₃ GX ₄ GL _2-3 ';

3) 5'-L ₁ -X ₁ TX ₂ TX ₃ TTGCX ₄ TL ₂ -3'.

In some embodiments, the one or more oligonucleotide sequences comprise 4N active test points, the 4N active test points are formed by each monomer mixture of the N monomer mixtures and 4 It is formed by the reaction of nucleotide coupling of different bases. specific,

4N active test points belong to the specific conjugation active test points. In some embodiments, further, the one or more oligonucleotide sequences comprise 4N active test points, and the 4N active test points are respectively different from 4 by each of the N monomer mixtures Coupling of bases is formed by the reaction of the test nucleotide coupling; numbering from 1 to 4 for the 4 coupling test nucleotides, and numbering from 1 to N for the N monomer mixture; the one or more oligonuclei Each oligonucleotide sequence in the nucleotide sequence can have the following structure:

5'-L ₁ -M[X _i Y _j ]-L ₂ -3'.

M is the number of sequence units containing Xi and Yj in the test region, M is an integer and 1≤M≤4N, the presence or absence of a spacer sequence comprising at least one nucleotide between adjacent sequence units;

Yj is the conjugated test nucleotide, j represents the number of the conjugated test nucleotide, and j=1, 2, 3, 4.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset The 2 oligonucleotide sequence information of the synthetic oligonucleotide chain, the 2 oligonucleotide sequences are set with 16 incorporation sites, and each monomer mixture has corresponding 4 incorporation sites, so that 2 The oligonucleotide sequence includes 16 active test points, 16 active test points belong to specific coupling active test points, and 16 active test points are composed of 4 monomer mixtures with adenine deoxynucleotide, guanine deoxynucleoside respectively. formed by the coupling of acid, cytosine deoxynucleotide and thymidine deoxynucleotide; the two oligonucleotide sequences can be respectively:

1) 5'-L ₁ -X ₁ TX ₂ TX ₁ GX ₂ GX ₃ AX ₄ AX ₃ CX ₄ CL _2-3 ';

2) 5'-L ₁ -X ₃ TX ₄ TX ₃ GX ₄ GX ₁ AX ₂ AX ₁ CX ₂ CL _2-3 '.

For example, selecting one of the 5 triplet nucleoside phosphoramidites as the internal standard and the remaining 4 as the test samples to prepare 4 monomer mixtures X ₁ , X ₂ , X ₃ and X ₄ , which can be based on preset 8 oligonucleotide sequence information to synthesize oligonucleotide chains, 8 oligonucleotide sequences are set with 16 incorporation sites, and each monomer mixture has corresponding 4 incorporation sites, so that 8 The oligonucleotide sequence includes 16 active test points, 16 active test points belong to specific coupling active test points, and 16 active test points are composed of 4 monomer mixtures with adenine deoxynucleotide, guanine deoxynucleoside respectively. formed by the coupling of acid, cytosine deoxynucleotide and thymidine deoxynucleotide; the 8 oligonucleotide sequences can be respectively:

1) 5'-L ₁ -X ₁ TCX ₁ AG-L _2-3 ';

2) 5'-L ₁ -X ₁ CAX ₁ GT-L _2-3 ';

3) 5'-L ₁ -X ₂ TCX ₂ AG-L ₂ -3';

4) 5'-L ₁ -X ₂ CAX ₂ GT-L _2-3 ';

5) 5'-L ₁ -X ₃ TCX ₃ AG-L ₂ -3';

6) 5'-L ₁ -X ₃ CAX ₃ GT-L _2-3 ';

7) 5'-L ₁ -X ₄ TCX ₄ AG-L ₂ -3';

8) 5'-L ₁ -X ₄ CAX ₄ GT-L ₂ -3'.

In some embodiments, further, the L1 comprises a binding site of a PCR-amplified forward primer, and the L2 comprises a binding site of a PCR-amplified reverse primer. Specifically, L1 and L2 can be combined with the forward primer and reverse primer of PCR amplification, respectively, so as to facilitate PCR amplification during subsequent sequencing. In some embodiments, the sequence of L1 can be SEQ ID NO:1. In some embodiments, the sequence of L2 can be SEQ ID NO:2.

In some embodiments, using the monomer mixture to synthesize the oligonucleotide chain further comprises: synthesizing the oligonucleotide chain by a method of solid phase synthesis. Preferably, in some embodiments, using the monomer mixture to synthesize the oligonucleotide chain further comprises: using a solid-phase synthesis method to synthesize the oligonucleotide chain in a DNA synthesizer.

In some embodiments, using the monomer mixture to synthesize the oligonucleotide chain further includes: obtaining a crude oligonucleotide after synthesizing the oligonucleotide chain, and separating and purifying the crude oligonucleotide to obtain a synthesized oligonucleotide chain. Specifically, by separating and purifying the crude oligonucleotide, the failed sequence of synthesis can be separated from the synthesized oligonucleotide chain, and a synthetic oligonucleotide chain with higher purity, that is, the pure oligonucleotide, can be obtained. In some embodiments, the crude oligonucleotide is separated and purified by one of the following processing methods: OPC purification method (Oligonucleotide Purification Cartridge, OPC), polyacylamide gel electrophoresis method (Polyacylamide Gel Electrophoresis, PAGE), Desalting purification method or high performance liquid chromatography (High Performance Liquid Chromatography, HPLC). The above separation and purification processing method can separate the synthesized failed sequence from the synthesized oligonucleotide chain, obtain pure oligonucleotide, and reduce the workload of sequencing statistics.

Step iv, oligonucleotide chain sequencing.

In some embodiments, sequencing the oligonucleotide chain comprises: sequencing the oligonucleotide chain synthesized in step iii. In some embodiments, in order to improve the sequencing speed and reduce the difficulty of sequencing, the oligonucleotide chain sequencing further includes using next generation sequencing technology (Next Generation Sequencing, NGS) to sequence the oligonucleotide chain synthesized in step iii. For example, synthetic oligonucleotide strands are sequenced using one of the following sequencing platforms: Illumina Hiseq sequencing platform, Illumina Miseq sequencing platform, Roche 454 sequencing platform, Life Technologies' Ion Torrent sequencing platform.

In step ⅴ, the relative reactivity ratio is determined based on the sequencing result.

In some embodiments, determining the relative reactivity ratio based on the sequencing result includes: counting the reads of the internal standard product and the reads of the test product corresponding to the at least N activity test points in the sequencing result respectively, and the internal standard substance of each activity test point is counted. The ratio of the reading to the analyte reading is the relative reactivity ratio of the internal standard to the analyte corresponding to the activity test point.

For example, the sequencing result can be the internal standard readings and the readings of the test product corresponding to N active test points respectively, and the N active test points belong to non-specific coupling activity test points; The ratio of the readings of the test product is the relative reactivity ratio of the internal standard product to the test product corresponding to the active test point. Relative reactivity with the corresponding analyte.

For example, the sequencing result can be the internal standard readings and the readings of the test product corresponding to N active test points respectively, and the N active test points belong to a specific coupling activity test point; The ratio of the product readings is the relative reactivity ratio of the internal standard product to the analyte corresponding to the active test point, and the relative reactivity ratio is reflected in the nucleotides (nucleosides with 4 different bases) that are coupled to a specific base. The relative reactivity of the internal standard product and the corresponding test product under the condition of any one of the acid). Each analyte has a corresponding relative reactivity ratio.

For example, the sequencing result can be the readings of the internal standard product and the sample to be tested corresponding to 2N active test points respectively, and the 2N active test points belong to a specific conjugated active test point; The ratio of the product readings is the relative reactivity ratio of the internal standard product to the analyte corresponding to the active test point, and the relative reactivity ratio is reflected in the nucleotides (nucleosides with 4 different bases) that are coupled to a specific base. The relative reactivity of the internal standard product and the corresponding test product under the conditions of any two of the acids). Each analyte has a corresponding 2 relative reactivity ratios.

For example, the sequencing result can be the internal standard readings and the readings of the test product corresponding to 3N active test points respectively, and the 3N active test points belong to a specific conjugated active test point; The ratio of the product readings is the relative reactivity ratio of the internal standard product to the analyte corresponding to the active test point, and the relative reactivity ratio is reflected in the nucleotides (nucleosides with 4 different bases) that are coupled to a specific base. The relative reactivity of the internal standard product and the corresponding test product under the conditions of any three of the acids). Each analyte has a corresponding 3 relative reactivity ratios.

For example, the sequencing result can be the readings of the internal standard product and the sample to be tested corresponding to 4N active test points respectively, and the 4N active test points belong to a specific conjugated active test point; The ratio of the product readings is the relative reactivity ratio of the internal standard product to the analyte corresponding to the active test point, and the relative reactivity ratio is reflected in the nucleotides (nucleosides with 4 different bases) that are coupled to a specific base. Under the condition of acid), the relative reactivity of the internal standard product and the corresponding test product. Each analyte has a corresponding 4 relative reactivity ratios.

In some embodiments, the above method further comprises: step iv, determining the relative reactivity coefficient of each test sample based on the relative reactivity ratio. In some embodiments, the one or more oligonucleotide sequences include N activity test points, and the relative reactivity coefficient of each analyte is the ratio of the relative reactivity of the internal standard to the corresponding analyte. In some embodiments, the one or more oligonucleotide sequences include 2N active test points, and the 2N active test points belong to a specific coupling activity test point, and each analyte has corresponding 2 relative responses Activity ratio, the relative reactivity coefficient of each test product is the average of two relative reactivity ratios between the internal standard and the corresponding test product. In some embodiments, the one or more oligonucleotide sequences include 3N active test points, and the 3N active test points belong to a specific conjugated active test point, and each analyte has corresponding 3 relative responses Activity ratio, the relative reactivity coefficient of each test product is the average of the three relative reactivity ratios of the internal standard product and the corresponding test product. In some embodiments, the one or more oligonucleotide sequences comprise 4N active test points, and the 4N active test points belong to a specific coupling activity test point, and each test sample has corresponding 4 relative responses Activity ratio, the relative reactivity coefficient of each test product is the average value of 4 relative reactivity ratios between the internal standard product and the corresponding test product. Specifically, each sample to be tested has a corresponding relative reactivity ratio measured by conjugated adenine deoxynucleotide, a relative reactivity ratio measured by conjugated guanine deoxynucleotide, and a corresponding relative reactivity ratio measured by conjugated cytosine deoxynucleotide. The relative reactivity ratio of a nucleotide assay, and the relative reactivity ratio of a conjugated thymidine deoxynucleotide assay, the relative reactivity coefficient of each test item is the average value of the corresponding four relative reactivity ratios above. Using the average value of the relative reactivity ratio to characterize the relative reactivity of each tripartite nucleoside phosphoramidite has higher accuracy and applicability.

According to the design method of an oligonucleotide synthesis reaction disclosed in the embodiments of this specification, the design method includes:

Specifically, the ratio of trinucleotide phosphoramidites, such as molar ratio, can be determined according to the relative reactivity ratio, which can adjust the distribution of each trinucleotide in the synthesized oligonucleotide chain. Further, when the library is established, it can be adjusted. Adjust mutation rate.

In some embodiments, determining the relative reactivity ratio of the internal standard to each test article comprises:

sequencing the synthesized oligonucleotide chain, and

Specifically, details on determining the relative reactivity ratio of the internal standard to each test sample can be found elsewhere in this disclosure.

According to a method for designing an oligonucleotide synthesis reaction disclosed in the embodiments of this specification, the design method includes: selecting AAC phosphoramidite among 20 triplet nucleoside phosphoramidites representing canonical amino acid codons as an internal standard , and the remaining 19 are the samples to be tested. Determine the relative reactivity coefficient of each test product based on the relative reactivity ratio of the internal standard product and each test product as shown in the table below, and determine according to the relative reactivity coefficient. Proportion of triple nucleoside phosphoramidites used in oligonucleotide synthesis reactions.

Specifically, each analyte has 4 relative reactivity ratios correspondingly determined under the condition of coupling nucleotides with 4 different bases, and the relative reactivity coefficient of each analyte is the ratio of the 4 relative reactivity ratios. average value. Details on determining the relative reactivity ratios and relative reactivity coefficients of the internal standard to each test article can be found elsewhere in this disclosure.

The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the embodiments do not limit the present invention. It should be understood that, for those skilled in the art, after understanding the principles of the present invention, various changes in form and details can be made in order to implement the present invention without departing from the principles.

The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.

Example 1. Selecting the internal standard product and the product to be tested, and preparing the mixed monomer

This example determines the relative reactivity of 20 triplet nucleoside phosphoramidites representing canonical amino acid codons.

1.1 AAC phosphoramidite was selected as the internal standard for the other 19 triple nucleoside phosphoramidites, and the other 19 triple nucleoside phosphoramidites were used as the test substance.

1.2 The 19 samples to be tested are mixed with the internal standard product in equal proportions to form 19 monomer mixtures, each monomer mixture contains the internal standard product and one sample to be tested.

Example 2. Synthesis of Oligonucleotide Chains

In this example, the oligonucleotide chain is synthesized on an automated DNA synthesizer, and the synthesis method is a solid-phase synthesis method. The model of the automated DNA synthesizer used in this example is Dr. Oligo48.

2.1 Dissolve adenosine phosphoramidite, guanosine phosphoramidite, cytosine nucleoside phosphoramidite, and thymidine phosphoramidite in acetonitrile at a concentration of 0.06M and place them in the automatic DNA synthesizer. In the corresponding channel; 19 monomer mixtures were respectively dissolved in a mixed solution of acetonitrile and dichloromethane (volume ratio=1:1) with a concentration of 0.15M, and were placed in the corresponding channels of the automated DNA synthesizer.

2.2 The information of the preset 20 oligonucleotide sequences, the preset oligonucleotide sequence information and the monomer mixture information are shown in Table 2 for details, and the information of the 20 oligonucleotide sequences is uploaded into the automated DNA synthesizer.

Table 2. Preset oligonucleotide sequence information and monomer mixture information table

Wherein: L1 is: 5'-CGGCAGCACATGTAGTGCAAGTCAAGGTT-3' (SEQ ID NO: 1); L2 is: 5'-ACCACTACTACTACACGCCGCTCACTCAT-3' (SEQ ID NO: 2).

2.3 Based on the information of the preset 20 oligonucleotide sequences, oligonucleotide chains were synthesized by solid-phase synthesis method (Beaucage et al., J. Tetrahedron Letters. 22.20:1859-1862 (1981)), and 20 groups were obtained Crude oligonucleotide; wherein, the coupling time of the monomer mixture is 300s×2 times.

Taking a group of oligonucleotide chains synthesized based on the oligonucleotide sequence Seq5 as an example, the group of oligonucleotide chains contains at least 2 or ³ kinds of nucleotide sequences. The specific sequence of the group of oligonucleotide chains See Table 3 for information.

Table 3. Nucleotide sequence information table

编号Numbering	核苷酸排列顺序(5’-3’)Nucleotide sequence (5'-3')
11	L1-CTGTTGGTGCTT-L2L1-CTGTTGGTGCTT-L2
22	L1-CTGTAACTGCTT-L2L1-CTGTAACTGCTT-L2
33	L1-CTGTTGGTAACT-L2L1-CTGTTGGTAACT-L2
44	L1-AACTTGGTGCTT-L2L1-AACTTGGTGCTT-L2
55	L1-AACTAACTGCTT-L2L1-AACTAACTGCTT-L2
66	L1-AACTTGGTAACT-L2L1-AACTTGGTAACT-L2
77	L1-CTGTAACTAACT-L2L1-CTGTAACTAACT-L2
88	L1-AACTAACTAACT-L2L1-AACTAACTAACT-L2

2.4 Perform HPLC gradient elution on the obtained 20 groups of crude oligonucleotides. The conditions of gradient elution are shown in Table 4; 20 groups of pure oligonucleotides were subjected to HPLC gradient elution, and the conditions of gradient elution are shown in Table 5. The 20 groups of pure oligonucleotides were based on the preset oligonucleotides, respectively. The nucleotide sequences Seq1-Seq20 were synthesized and purified. The HPLC analysis patterns of the 20 groups of pure oligonucleotides are shown in Figures 1 to 20, and the HPLC analysis and purity of the 20 groups of pure oligonucleotides are shown in Table 6.

Table 4. Gradient elution reaction conditions for crude oligonucleotides

流动相mobile phase	浓度％concentration%	时间mintime min
乙腈Acetonitrile	8-188-18	0-150-15
乙腈Acetonitrile	8888	15.01-1715.01-17
乙腈Acetonitrile	88	17.01-1917.01-19

Table 5. Gradient elution reaction conditions for pure oligonucleotides

流动相mobile phase	浓度％concentration%	时间mintime min
乙腈Acetonitrile	8-188-18	0-150-15
乙腈Acetonitrile	8888	15.01-1715.01-17

流动相mobile phase	浓度％concentration%	时间mintime min
乙腈Acetonitrile	88	17.01-1917.01-19

Table 6. HPLC determination of purity of 20 groups of oligonucleotides

Example 3. Oligonucleotide library construction and next-generation sequencing

3.1 Building a library

3.1.1 Design amplification primers

The sequence of the designed forward primer is: 5'-TCGTGTCAAGTACGGCAGCA-3' (SEQ ID NO: 3); the sequence of the designed reverse primer is: 5'-CCAGACCCGATATGAGTGAGC-3' (SEQ ID NO: 4). Among them, the forward primer contains a binding site with L1, and the reverse primer contains a binding site with L2.

3.1.2 Polymerase chain reaction (PCR) for library construction

The PCR reaction system is shown in Table 7.

Table 7. PCR reaction system table

组分component	体积量μLvolume μL
单链模板(150ng)Single stranded template (150ng)	55
正向引物(10uM)Forward primer (10uM)	11
反向引物(10uM)Reverse primer (10uM)	11
预混液premix	2525
无核酸酶水Nuclease-free water	1818

The synthetic oligonucleotide chain obtained in Example 3 was added as a single-stranded template to a PCR reaction system configured as shown in Table 7 and placed in a PCR reaction tube.

Place the PCR reaction tube loaded with the PCR reaction system on the PCR machine, set up and run the PCR program shown in Table 8, and obtain PCR products.

Table 8. PCR program description

3.1.3 Magnetic bead purification

Use 110 μL of Yeasen Magnetic Bead Kit (Hieff NGS DNA Separation Magnetic Beads produced by Shanghai Yisheng Biotechnology Co., Ltd., Cat. No. 12599ES03) to purify the PCR product, and take 20 μL of the eluate in the Yeasen Magnetic Bead Kit for elution, 18 μL of purified PCR product was recovered. 18 μL of purified PCR product contained 300 ng of purified oligonucleotide strands.

3.2 Connecting to build a library

3.2.1 End Repair

The end repair reaction mixture was prepared according to Table 9, wherein the purified oligonucleotide strands were added as DNA inserts to the end repair reaction mixture.

Place the end-repair reaction mixture configured as shown in Table 9 into a PCR reaction tube.

Table 9. Proportion information of end repair reaction mixture

试剂名称Reagent name	体积(μL)Volume (μL)
DNA插入片断(200ng)DNA insert (200ng)	3030
末端修复缓冲液end repair buffer	17.817.8
末端修复酶end repair enzyme	2.202.20
总体积 total capacity	5050

Place the PCR reaction tube loaded with the end-repair reaction mixture on the PCR machine, set and run the PCR program shown in Table 10, and obtain the end-repair product.

Table 10. PCR instrument operating conditions table

温度temperature	时间time	循环数number of cycles
37℃37℃		30min30min	11
75℃75℃		30min30min	11
4℃4℃	维持至下一步keep to the next step	11

3.2.2 Sequencing adapter ligation

Adapter ligation was performed using the next-generation sequencing library preparation kit KAPA HyperPlus kit (manufactured by Roche Diagnostics (Shanghai) Co., Ltd., Cat. No. KK8512).

Configure the sequencing adapter ligation reaction mix according to Table 11.

Table 11. Sequencing adapter ligation mix configuration table

试剂名称Reagent name	体积(μL)Volume (μL)
连接酶缓冲液Ligase Buffer	27.7527.75
Ligase Enzyme连接酶 Ligase Enzyme Ligase	11
总体积total capacity	28.7528.75

Put the PCR reaction tube containing the end repair products on ice, add 1.25 μL of sequencing adapters (selected from D501-D508 adapters and D701-D712 adapters in Illumina TruSeq HT Kits) and 28.75 μL of sequencing adapters into the PCR reaction tube The connector is connected to the mixture.

Place the PCR reaction tube loaded with the end repair product, sequencing adapter ligation mixture and sequencing adapter on the PCR machine, set and run the PCR program shown in Table 12, and obtain the sequencing adapter ligation product.

Table 12. PCR instrument operating conditions table

温度temperature	时间time	循环数number of cycles
23℃23℃		15min15min	11
4℃4℃	维持至下一步keep to the next step	11

3.2.3 Magnetic bead purification

Use 110 μL of Yeasen Magnetic Bead Kit (Hieff NGS DNA Separation Magnetic Beads produced by Shanghai Yisheng Biotechnology Co., Ltd., Cat. No. 12599ES03) to purify the sequencing adapter ligation product, and take 16 μL of the eluate in the Yeasen Magnetic Bead Kit for washing 15 μL of purified sequencing adapter ligation product was recovered.

3.2.4 Next-generation sequencing and statistical analysis

Next-generation sequencing was performed on the purified sequencing adapter ligation product to obtain the reads corresponding to the analyte sequence and the internal standard sequence of 76 active test points. The relative reactivity ratio of the internal standard substance and the test substance, the relative reactivity ratio reflects the relative reactivity ratio of the internal standard substance and the test substance under the conditions of coupling nucleotides with specific bases.

Taking X ₄ as an example, the preset 4 oligonucleotide sequences Seq5-Seq8 respectively comprise active test points X ₄ A, which are formed by the conjugated adenine deoxynucleotide corresponding to the monomer mixture X ₄ (AAC/CTG). Active test site X ₄ G formed by coupling guanine deoxynucleotide, active test site X ₄ C formed by coupling cytosine deoxynucleotide, and active test site X ₄ T formed by coupling thymidine deoxynucleotide . The oligonucleotide chain synthesized based on the predetermined oligonucleotide sequence Seq5-8 contains 4 sets of oligonucleotide sequences, and the sequencing result of a set of oligonucleotide sequences generated based on the corresponding oligonucleotide sequence Seq5 can be read Take the reading of the internal standard product and the reading of the test product at the active test point X ₄ T of the CTG phosphoramidite. Based on the sequencing results of a group of oligonucleotide sequences generated by the corresponding oligonucleotide sequence Seq6, the readings of the internal standard product and the reading of the test product of the active test point X ₄ C of the CTG phosphoramidite can be read. Based on the sequencing results of a group of oligonucleotide sequences generated by the oligonucleotide sequence Seq7, the internal standard reading and the reading of the test product of the active test point X ₄ G of the CTG phosphoramidite can be read. Corresponding to the sequencing results of a group of oligonucleotide sequences generated by the oligonucleotide sequence Seq8, the readings of the internal standard product and the reading of the test product of the active test point X ₄ A of the CTG phosphoramidite can be read. The specific readings and relative reactivity ratios are shown in Table 13.

Table 13. Active Test Point Readout Table for CTG Phosphoramidite

读取数number of reads	AACAAC	CTGCTG	相对反应活性比Relative reactivity ratio
TT	71627162	40174017	1.781.78
CC	56945694	19711971	2.892.89
GG	39753975	19931993	1.991.99
AA	56005600	33943394	1.651.65

The results of next-generation sequencing and statistical analysis are shown in Table 14.

Table 14. Relative Reactivity Coefficients of Different Trinucleoside Phosphoramidites and Internal Standard AAC

Note: The relative reactivity ratio and relative reactivity coefficient of the internal standard AAC phosphoramidite are all 1.

It can be seen from Table 14 that the reactivity of 19 triplet nucleoside phosphoramidites relative to AAC phosphoramidites can be divided into three categories, and there are 5 monomers with higher reactivity than AAC, and the reactivity coefficients are between 0.8 and 0.9; There are 8 kinds of monomers with comparable reactivity of AAC, and the reactivity coefficients are between 1.0-1.4; there are 6 kinds of monomers with lower reactivity than AAC, and the reactivity coefficients are between 1.4-2.1.

Embodiment 4, verify the relative reactivity of triple nucleoside phosphoramidites

In order to verify the data accuracy of the measured relative reactivity coefficients, 20 triplet nucleoside phosphoramidites representing canonical amino acid codons were prepared into mixed monomers X ₂₀ with a ratio of 5% according to the relative reactivity data, and the mixed monomers were mixed. Monomer _{X20 is incorporated into the} oligonucleotide sequence. Considering the different reactivity of the 5'-terminal hydroxyl groups of the four different nucleosides with the trinucleoside phosphoramidite reaction substrates, the oligonucleotide sequence Seq21 was designed, which contains mixed monomers with Activity test point formed by nucleotide coupling of 4 different bases. The specific sequence information of the oligonucleotide sequence Seq21 is:

5'-L1-X ₂₀ TX ₂₀ GX ₂₀ CX ₂₀ A-L2-3'

Wherein, L1 is: 5'-CGGCAGCACATGTAGTGCAAGTCAAGGTT-3' (SEQ ID NO: 1);

L2 is 5'-ACCACTACTACTACACGCCGCTCACTCAT-3' (SEQ ID NO: 2).

Synthesis, library construction and next-generation sequencing of oligonucleotide chains were carried out with reference to Example 2 and Example 3. Based on the preset oligonucleotide sequence Seq21 information, the crude oligonucleotide is synthesized, and the crude oligonucleotide is separated and purified to obtain the pure oligonucleotide, which is the synthetic oligonucleotide chain. The HPLC analysis pattern of the pure oligonucleotide is shown in Figure 21, and the purity of the pure oligonucleotide determined by HPLC is 99.6%. The results of the sequencing statistical analysis of the synthesized oligonucleotide chains are shown in Table 14. As can be seen from Table 14, in a group of oligonucleotide chains synthesized based on the oligonucleotide sequence Seq21, the proportions of the 20 codons in each different activity test point of the oligonucleotide chain are all within the preset range ( 3.5-6.5%), this ratio range is accurate enough for downstream applications such as library construction; in addition, the standard deviation of the ratio of the same codon measured at the activity test points corresponding to four different nucleosides is 0.001- 0.009, which proves the reliability of the above reactivity coefficient.

Table 14. The proportion of 20 codons in different activity test points of oligonucleotide chains

Those skilled in the art should understand that the above embodiments are only to illustrate the present invention, but not to limit the present invention. Any modifications, equivalent substitutions and changes made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

A method for measuring the relative reactivity of triple nucleoside phosphoramidites, comprising:

ⅰ) Select any one of N+1 different triple nucleoside phosphoramidites as the internal standard, and the remaining N triple nucleoside phosphoramidites as the test sample, where N is an integer and N≥1;

ii) Prepare N monomer mixtures, wherein each of the monomer mixtures includes the internal standard product and one of the test products, and the internal standard product and the test product in each of the monomer mixtures The molar ratios of the products are the same, and the test products in each of the monomer mixtures are different from each other;

iii) synthesizing an oligonucleotide chain based on the preset sequence information of one or more oligonucleotides to obtain a synthetic oligonucleotide chain, wherein, by setting the incorporation sites of the N monomer mixtures, causing the one or more oligonucleotide sequences to include at least N active test points, each of the monomer mixtures having a corresponding at least one active test point;

iv) sequencing the synthesized oligonucleotide strand, and

ⅴ) Determine the relative reactivity ratio of the internal standard product to each test product based on the sequencing results.
The method for determining the relative reactivity of triple nucleoside phosphoramidites according to claim 1, wherein the oligonucleotide sequence has a test area and a non-test area, and the at least N active test points are distributed in the The test region of the one or more oligonucleotide sequences, and the independent presence or absence of a spacer sequence comprising at least one nucleotide between any two adjacent active test sites.
The method for determining the relative reactivity of triple nucleoside phosphoramidites according to claim 1, wherein the one or more oligonucleotide sequences comprise N activity test points, and the N activity test points are composed of Each of the N monomer mixtures is randomly incorporated into a test region of the one or more oligonucleotide sequences formed by reaction; or, the one or more oligonucleotide sequences include N active test sites, the N active test sites are formed by reacting each of the N monomer mixtures with a nucleotide coupling of the same base, respectively.
The method for determining the relative reactivity of a tripartite nucleoside phosphoramidite according to claim 1, wherein the one or more oligonucleotide sequences comprise 2N activity test points, and the 2N activity test points are composed of Each of the N monomer mixtures is formed by reacting with nucleotide couplings of two different bases, respectively.
The method for determining the relative reactivity of a tripartite nucleoside phosphoramidite according to claim 1, wherein the one or more oligonucleotide sequences comprise 3N activity test points, and the 3N activity test points are composed of Each of the N monomer mixtures is formed by reacting with nucleotide couplings of 3 different bases, respectively.
The method for determining the relative reactivity of triple nucleoside phosphoramidites according to claim 1, wherein the one or more oligonucleotide sequences comprise 4N activity test points, and the 4N activity test points are composed of Each of the N monomer mixtures is formed by reacting with nucleotide couplings of 4 different bases, respectively.
The method for determining the relative reactivity of a triplet nucleoside phosphoramidite according to any one of claims 1 to 6, wherein any one triplet nucleoside in the N+1 triplet nucleoside phosphoramidites The phosphoramidites all correspond to a codon encoding an amino acid, and the codons corresponding to any two triple nucleoside phosphoramidites in the N+1 triple nucleoside phosphoramidites encode different amino acids.
The method for determining the relative reactivity of a tripartite nucleoside phosphoramidite according to claim 7, wherein the N is any positive integer within 18.
The method for determining the relative reactivity of trinucleoside phosphoramidites according to claim 7, wherein the N is 19.
The method for determining the relative reactivity of triple nucleoside phosphoramidites according to claim 7, wherein the selected internal standard is AAC phosphoramidites.
The method for measuring the relative reactivity of triple nucleoside phosphoramidite according to claim 1, wherein the determining the relative reactivity ratio of the internal standard substance and each test substance based on the sequencing result comprises: counting all the reactivity in the sequencing result. The internal standard reading and the reading of the product to be tested are respectively corresponding to the at least N active test points, and the ratio of the reading of the internal standard to the reading of each active test point is the internal standard and the corresponding to the active test point. The relative reactivity ratio of the product.
The method for determining the relative reactivity of trinucleoside phosphoramidites according to claim 11, wherein the method further comprises: vi) determining the relative reactivity coefficient of each test sample based on the relative reactivity ratio.
The method for determining the relative reactivity of trinucleoside phosphoramidites according to claim 1, wherein the molar ratio of the internal standard substance to the test substance in each monomer mixture is 1:1.
The method for determining the relative reactivity of triple nucleoside phosphoramidites according to claim 1, wherein the sequencing of the synthesized oligonucleotide chain comprises: using next generation sequencing technology to synthesize the oligonucleotide Strand sequencing.
The method for determining the relative reactivity of tripartite nucleoside phosphoramidites according to claim 1, wherein the oligonucleotide chain synthesis based on the preset one or more oligonucleotide sequence information comprises: in In a DNA synthesizer, the solid-phase synthesis method is used for oligonucleotide chain synthesis.
The application of the relative reactivity of triple nucleoside phosphoramidite measured by the method according to any one of claims 1 to 15 in constructing a gene library.
A method for designing an oligonucleotide synthesis reaction, comprising:

ⅰ) Select any one of N+1 different triple nucleoside phosphoramidites as the internal standard, and the remaining N triple nucleoside phosphoramidites as the test samples, where N is an integer and N≥1;

ii) determining the relative reactivity ratio of the internal standard to each test article; and

iii) Determine the ratio of triple nucleoside phosphoramidites used in the reaction of synthesizing oligonucleotides according to the relative reactivity ratio of the internal standard substance and each test substance.
The method for designing an oligonucleotide synthesis reaction according to claim 17, wherein determining the relative reactivity ratio of the internal standard substance to each test substance comprises:

Prepare N monomer mixtures, wherein each of the monomer mixtures includes the internal standard and one of the samples to be tested, and the internal standard and the sample to be tested in each of the monomer mixtures are different from each other. The molar ratios are the same, and the samples to be tested in each of the monomer mixtures are different from each other;

Perform oligonucleotide chain synthesis based on preset one or more oligonucleotide sequence information to obtain a synthesized oligonucleotide chain, wherein by setting the incorporation sites of the N monomer mixtures, all The one or more oligonucleotide sequences include at least N activity test points, and each of the monomer mixtures has a corresponding at least one activity test point;

sequencing the synthesized oligonucleotide chain, and

Based on the sequencing results, the relative reactivity ratio of the internal standard product to each test product was determined.
The method for designing an oligonucleotide synthesis reaction according to claim 17, wherein,

The AAC phosphoramidite among the 20 triple nucleoside phosphoramidites representing canonical amino acid codons was selected as the internal standard, and the remaining AAA phosphoramidites, ACT phosphoramidites, ATC phosphoramidites, ATG phosphoramidites, CAG Phosphoramidite, CAT phosphoramidite, CCG phosphoramidite, CGT phosphoramidite, CTG phosphoramidite, GAA phosphoramidite, GAC phosphoramidite, GCT phosphoramidite, GGT phosphoramidite, GTT phosphoramidite, TAC Phosphoramidites, TCT phosphoramidites, TGC phosphoramidites, TGG phosphoramidites and TTC phosphoramidites are the samples to be tested.
A method for designing an oligonucleotide synthesis reaction, comprising: selecting AAC phosphoramidites in 20 triplet nucleoside phosphoramidites representing canonical amino acid codons as an internal standard, and the remaining 19 are to be tested Determine the relative reactivity coefficient of each test product based on the relative reactivity ratio of the internal standard product and each test product as shown in the following table, and determine the relative reactivity coefficient of the synthetic oligonucleotide according to the relative reactivity coefficient. Proportion of triple nucleoside phosphoramidite used.
The relative reactivity coefficient of triple nucleoside phosphoramidites, which is characterized in that AAC phosphoramidite in 20 triple nucleoside phosphoramidites representing canonical amino acid codons is selected as the internal standard, and the remaining 19 are the samples to be tested , and determine the relative reactivity coefficient of each test product calculated based on the relative reactivity ratio of the internal standard product and each test product as shown in the following table.
The application of the relative reactivity coefficient of the triple nucleoside phosphoramidite according to claim 21 in the construction of a gene library.