Imaging reagents, kits for inhibiting reduction in fluorescence intensity

Abstract

The present application relates to the technical field of gene sequencing, in particular to imaging reagent for inhibiting fluorescence intensity reduction and kit. In the imaging reagent, 3,4-dihydroxybenzoic acid ethyl ester, 3,4-dihydroxybenzoic acid and sodium ascorbate are compounded and synergistically enhanced, so that the generation of free radicals and active oxygen clusters in the process of fluorescent molecular imaging is reduced, guanine oxidation caused by free radicals and / or active oxygen cluster molecules is reduced, or the homodimerization and easy dimerization of thymine and cytosine at the dealkylated site are reduced, nucleic acid damage is reduced, the reduction of fluorescence intensity in the sequencing process is reduced, the fluorescence intensity is maintained within a small range in the long read sequencing process, the phasing and predetermination are reduced, and the accuracy of long read sequencing is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene sequencing technology, in particular to an imaging reagent for inhibiting the reduction of fluorescence intensity and a kit. BACKGROUND

[0002] As an important experimental technique, gene sequencing has been widely used in biological research. As soon as the DNA double helix structure was discovered, DNA sequencing technology was reported, but the operation process was complex and could not be scaled up. DNA sequencing technology has developed from the first-generation sequencing technology represented by the Sanger method to the second-generation sequencing technology of sequencing by synthesis, and then to the third-generation sequencing technology represented by single molecule sequencing technology such as nanopore. The second-generation sequencing technology of sequencing by synthesis includes forming a template cluster structure in an array structure, using different colors of fluorescent labels to mark four different dNTPs, and releasing different fluorescence when a DNA polymerase synthesizes a complementary strand by adding a dNTP. According to the captured fluorescence signal and through specific computer software processing, the sequence information of the DNA to be tested is obtained.

[0003] The repeated fluorescence detection steps in the array case of the sequencing by synthesis detection method can cause nucleic acid damage and result in the loss of fluorescence intensity. The energy transition of fluorescent molecules after absorbing excitation light, and the retransformation of excited state to ground state, usually accompanied by the generation of oxygen free radicals, forming reactive oxygen cluster molecules. Under the action of oxygen free radicals or reactive oxygen cluster molecules, guanine is converted to 8-oxo-guanine, or causes dealkylation sites and thymine and cytosine homodimerization and easy dimerization, ultimately leading to nucleic acid damage or degradation. Therefore, in order to reduce the loss of fluorescence intensity caused by nucleic acid damage in the fluorescence detection process, an antioxidant is usually added to the imaging reagent to remove oxygen free radicals and avoid the formation of reactive oxygen cluster molecules. For example, ascorbate is added in patent WO2006064199, and polyphenolic compounds such as gallic acid are added in patent CN103403188B. Although the addition of antioxidant components in these prior art imaging reagents can reduce the fluorescence intensity decay to some extent during sequencing, the loss of fluorescence intensity still exists with the increase of sequencing read length, resulting in the reduction of sequencing accuracy for long read sequencing. Therefore, how to further optimize the formula composition of the imaging reagent and further improve the accuracy of long read sequencing has become a technical problem to be solved. SUMMARY

[0004] In order to overcome the defects of the prior art, one of the purposes of the present application is to provide an imaging reagent for inhibiting the reduction of fluorescence intensity, which is applied to a multi-cycle nucleic acid sequencing reaction based on fluorescence. By compounding multiple antioxidants, a synergistic effect is achieved to inhibit the reduction of fluorescence intensity with the extension of sequencing read length and improve the sequencing accuracy.

[0005] Meanwhile, the present application also provides a kit comprising the imaging reagent provided by the present application, which is applied to nucleic acid sequencing to improve the accuracy of long read sequencing.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0007] An imaging reagent for inhibiting the reduction of fluorescence intensity, comprising 3,4-dihydroxybenzoic acid ethyl ester, 3,4-dihydroxybenzoic acid and sodium ascorbate.

[0008] Optionally, the concentration ratio of 3,4-dihydroxybenzoic acid ethyl ester, 3,4-dihydroxybenzoic acid and sodium ascorbate in the imaging reagent is 1:1:3. Further, the imaging reagent further comprises Tris buffer.

[0009] In the imaging reagent of the present application, 3,4-dihydroxybenzoic acid ethyl ester, 3,4-dihydroxybenzoic acid and sodium ascorbate are compounded and synergistically act together to reduce the generation of free radicals and reactive oxygen clusters in the process of fluorescent molecular imaging, reduce the guanine oxidation caused by free radicals and / or reactive oxygen cluster molecules, or the homodimerization and heterodimerization of thymine and cytosine at the abasic site, and reduce nucleic acid damage, thereby reducing the reduction of fluorescence intensity in the sequencing process, maintaining the fluorescence intensity within a small range of fluctuation in the long read sequencing process, reducing phasing and pre-phasing, and improving the accuracy of long read sequencing.

[0010] As a preferred embodiment, the specific formula of the imaging reagent comprises 90-110 mM Tris, 750-850 mM NaCl, 15-25 mM 3,4-dihydroxybenzoic acid ethyl ester, 15-25 mM 3,4-dihydroxybenzoic acid, 45-65 mM sodium ascorbate, 0.1%-0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the balance is water, and the pH is 8.0.

[0011] As a further preferred embodiment, in one embodiment of the present application, the formula of the imaging reagent comprises 100 mM Tris, 800 mM NaCl, 20 mM 3,4-dihydroxybenzoic acid ethyl ester, 20 mM 3,4-dihydroxybenzoic acid, 60 mM sodium ascorbate, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the balance is water, and the pH is 8.0.

[0012] A kit for nucleic acid sequencing, comprising an extension reagent, a cleavage reagent, an imaging reagent and a washing agent; wherein the extension reagent comprises modified nucleotides, and the modified nucleotide molecules are connected with a reversible blocking moiety; wherein the cleavage reagent comprises a component acting on the reversible blocking moiety to separate the reversible blocking moiety from the modified nucleotide molecules; and the imaging reagent is the imaging reagent described above.

[0013] Optionally, the modified nucleotide molecule is linked to a label molecule through a cleavable linker; the cleavable linker comprises one or more cleavable groups; the cleavable groups comprise disulfide bond and / or azide; the cleaving reagent further comprises a component acting on the cleavable groups to detach the label molecule from the modified nucleotide molecule; the reversible blocking moiety is methyl azide. As a further preferred, the label molecule is a fluorescent molecule.

[0014] Optionally, the washing solution comprises an antioxidant and / or a cleaving reagent scavenger.

[0015] Optionally, the cleavable group is azide; the cleaving reagent comprises tris-(hydroxyl propyl) phosphine; the washing solution comprises cystine, thioctic acid, 3,3'-dithiodipropionic acid or polyethyleneglycolated azide.

[0016] Optionally, the washing solution further comprises gallic acid and / or sodium ascorbate.

[0017] In one embodiment of the present application, the nucleic acid sequencing method using the kit of the present application comprises: 1) providing an array of nucleic acid template molecules; 2) adding sequencing primers to hybridize with the corresponding nucleic acid template molecules; 3) adding an extension reagent, under the action of polymerase in the extension reagent, modified nucleotide molecules are incorporated into the sequencing primers; 4) then adding a washing solution to remove unreacted extension reagent; 5) then adding an imaging reagent, under the action of excitation light, fluorescent molecules linked to the modified nucleotide molecules through a cleavable linker emit light, and an image of the fluorescent molecules emitting light of the array of nucleic acid template molecules is collected, and the type of modified nucleotide incorporated for each template molecule is determined according to the image information; the imaging reagent used in the present application comprises 3,4-dihydroxybenzoic acid ethyl ester, 3,4-dihydroxybenzoic acid and sodium ascorbate, and the three components have a synergistic effect to reduce the photo-induced damage of nucleic acid molecules in the sequencing process; 6) then adding a washing solution to replace and flush away the imaging reagent, and then adding a cleaving reagent to remove the reversible blocking moiety and the label molecule on the modified nucleotide molecules incorporated into the sequencing primers; 7) then adding a washing solution to replace and flush away unreacted cleaving reagent; the washing solution in this step can eliminate the oxygen free radicals generated in the reaction system by adding an antioxidant such as gallic acid and / or ascorbic acid, and at the same time, a cleaving reagent scavenger is added, which is a compound that inhibits the interaction between the reactive compounds used or generated in step 6) and the polynucleotides used in the sequencing process, the enzymes used in the sequencing process or other reagents or compounds used in the sequencing process, such as cystine, thioctic acid, 3,3'-dithiodipropionic acid or polyethyleneglycolated azide, which can oxidize the residual cleaving reagent to eliminate the influence of the residual cleaving reagent on the incorporation of the next round of nucleotides; 8) then returning to step 3) to cycle the sequencing. Attached Figure Description

[0018] Figure 1 This is a graph showing the trend of fluorescence intensity changes during sequencing using the Illumina NextSeq500 reagents.

[0019] Figure 2 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Example 1 of this invention.

[0020] Figure 3 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 1 of this invention.

[0021] Figure 4 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 2 of this invention.

[0022] Figure 5 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 3 of this invention.

[0023] Figure 6 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 4 of this invention.

[0024] Figure 7 This is a graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 5 of this invention.

[0025] Figure 8 A graph showing the trend of fluorescence intensity changes during sequencing using the imaging reagents provided in Comparative Example 6 of this invention. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available.

[0027] Example 1

[0028] This embodiment provides an imaging reagent to suppress fluorescence intensity reduction. Its formulation consists of 100mM Tris, 800mM NaCl, 20mM ethyl 3,4-dihydroxybenzoate, 20mM 3,4-dihydroxybenzoic acid, 60mM sodium ascorbate, 0.1wt% 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate, with the balance being water, and pH=8.0.

[0029] Its preparation method includes:

[0030] 1) Respectively weigh 3.0285 g of Tris, 11.688 g of NaCl, 2.9718 g of sodium ascorbate, 0.911 g of ethyl 3,4-dihydroxybenzoate, 0.7705 g of 3,4-dihydroxybenzoic acid, 0.25 g of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in a 250 mL beaker;

[0031] 2) Add 200 mL of ultrapure water to the beaker, put the rotor, and place the beaker on the magnetic stirrer to dissolve;

[0032] 3) After dissolving, adjust the pH to 8.0 using concentrated hydrochloric acid;

[0033] 4) Pour the solution into a graduated cylinder to make up to 250 mL, filter the reagent after making up, and store at -20°C.

[0034] Example 2

[0035] This example provides an imaging reagent that suppresses the decrease in fluorescence intensity, which has a formulation composition of 90 mM Tris, 750 mM NaCl, 15 mM ethyl 3,4-dihydroxybenzoate, 15 mM 3,4-dihydroxybenzoic acid, 45 mM sodium ascorbate, 0.1 wt% of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the balance is water, pH = 8.0. The preparation method process is the same as Example 1.

[0036] Example 3

[0037] This example provides an imaging reagent that suppresses the decrease in fluorescence intensity, which has a formulation composition of 110 mM Tris, 850 mM NaCl, 25 mM ethyl 3,4-dihydroxybenzoate, 25 mM 3,4-dihydroxybenzoic acid, 65 mM sodium ascorbate, 0.2 wt% of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the balance is water, pH = 8.0. The preparation method process is the same as Example 1.

[0038] Comparative Example 1

[0039] This comparative example provides an imaging reagent, which has a formulation composition of 100 mM Tris, 800 mM NaCl, 100 mM ethyl 3,4-dihydroxybenzoate, 0.1 wt% of 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the balance is water, pH = 8.0. The preparation method process is the same as Example 1.

[0040] Comparative Example 2

[0041] This comparative example provides an imaging reagent having a formulation consisting of 100 mM Tris, 800 mM NaCl, 100 mM 3,4-dihydroxybenzoic acid, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, the balance being water, pH = 8.0. The method of preparation is the same as Example 1.

[0042] Comparative Example 3

[0043] This comparative example provides an imaging reagent having a formulation consisting of 100 mM Tris, 800 mM NaCl, 100 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, the balance being water, pH = 8.0. The method of preparation is the same as Example 1.

[0044] Comparative Example 4

[0045] This comparative example provides an imaging reagent having a formulation consisting of 100 mM Tris, 800 mM NaCl, 50 mM ethyl 3,4-dihydroxybenzoate, 50 mM 3,4-dihydroxybenzoic acid, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, the balance being water, pH = 8.0. The method of preparation is the same as Example 1.

[0046] Comparative Example 5

[0047] This comparative example provides an imaging reagent having a formulation consisting of 100 mM Tris, 800 mM NaCl, 60 mM ethyl 3,4-dihydroxybenzoate, 20 mM 3,4-dihydroxybenzoic acid, 20 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, the balance being water, pH = 8.0. The method of preparation is the same as Example 1.

[0048] Comparative Example 6

[0049] This comparative example provides an imaging reagent having a formulation consisting of 100 mM Tris, 800 mM NaCl, 20 mM ethyl 3,4-dihydroxybenzoate, 60 mM 3,4-dihydroxybenzoic acid, 20 mM sodium ascorbate, 0.1 wt% 3-[(3-cholamidopropyl)dimethylammonio]-1 -propanesulfonate, the balance being water, pH = 8.0. The method of preparation is the same as Example 1.

[0050] Test Example

[0051] Test method: the present application uses Illumina NextSeq500 sequencer V2.1 to run the test, under the technical principle of sequencing by synthesis, the influence of the imaging reagent of the present application and the imaging reagent of the comparative example on the fluorescence signal in the sequencing reaction process, and the influence on the sequencing read length and accuracy are analyzed by comparative test, the specific test includes 150 cycles and 288 titles of phix genome;

[0052] Test grouping: the test is divided into 8 groups, wherein the first group uses the reagent matched with Illumina NextSeq500 sequencer; the second group uses the imaging reagent provided by example 1 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the third group uses the imaging reagent provided by comparative example 1 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the fourth group uses the imaging reagent provided by comparative example 2 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the fifth group uses the imaging reagent provided by comparative example 3 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the sixth group uses the imaging reagent provided by comparative example 4 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the seventh group uses the imaging reagent provided by comparative example 5 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer; the eighth group uses the imaging reagent provided by comparative example 6 to replace the imaging reagent in the reagent matched with Illumina NextSeq500 sequencer, and other reagents use the reagent matched with Illumina NextSeq500 sequencer.

[0053] Test results:

[0054] 1. The sequencing results of each group for 150 single-end cycles are shown in Table 1 below:

[0055] Table 1

[0056]

[0057] 2. The trends in fluorescence intensity during 150 single-end sequencing cycles in each group are as follows: Figures 1 to 8 As shown, where Figure 1 This graph shows the trend of fluorescence intensity changes during sequencing using commercially available reagents. Figure 2 This diagram shows the fluorescence intensity change trend during sequencing using the imaging reagent provided by this invention, which is composed of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, and sodium ascorbate. Figure 3 This graph shows the trend of fluorescence intensity changes when an imaging reagent composed solely of ethyl 3,4-dihydroxybenzoate is used during sequencing. Figure 4 This graph shows the trend of fluorescence intensity changes when an imaging reagent composed solely of 3,4-dihydroxybenzoic acid is used during sequencing. Figure 5 This graph shows the trend of fluorescence intensity changes when an imaging reagent consisting only of sodium ascorbate is used during sequencing. Figure 6 This graph shows the trend of fluorescence intensity changes during sequencing when an imaging reagent composed of ethyl 3,4-dihydroxybenzoate and 3,4-dihydroxybenzoic acid is applied. Figure 7 and Figure 8 All images show the trend of fluorescence intensity changes during sequencing when the imaging reagent, composed of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, and sodium ascorbate in a mixed ratio is applied.

[0058] Depend on Figures 1 to 8 As shown in Table 1, the imaging reagent of this invention, composed of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, and sodium ascorbate, achieves the same effect as the commercially available Illumina NextSeq500 imaging reagent in suppressing fluorescence intensity decay during sequencing reactions. It maintains overall stable fluorescence intensity throughout the sequencing process without significant attenuation. Furthermore, the data in Table 1 also show that the imaging reagent of this invention, composed of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, and sodium ascorbate, achieves the same level as commercially available products in terms of phasing value, Q30, and error rate, and can replace commercially available products.

[0059] The ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid alone as an imaging reagent applied in the sequencing reaction cannot inhibit the decay of the fluorescence intensity, a large number of phasing errors occur in the sequencing process, Q30 is lower than 30, the error rate of sequencing is more than 40, and it cannot be used as an imaging reagent for sequencing reaction; the imaging reagent composed of ascorbic acid alone or ethyl 3,4-dihydroxybenzoate and 3,4-dihydroxybenzoic acid, although compared with ethyl 3,4-dihydroxybenzoate and 3,4-dihydroxybenzoic acid, the decay amplitude of the fluorescence intensity is reduced as an imaging reagent applied in the sequencing reaction, but it still cannot maintain the overall stability of the fluorescence intensity during the whole sequencing reaction process, and the data shown in Table 1 also shows that the phasing value, Q30 and error rate of the imaging reagent composed of ascorbic acid alone or ethyl 3,4-dihydroxybenzoate and 3,4-dihydroxybenzoic acid cannot reach the level of the imaging reagent of the present application, so it can be seen that the ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid and ascorbic acid sodium can produce a synergistic effect when used in combination, and as an imaging reagent applied in the sequencing reaction, it can inhibit the fluorescence damage and improve the sequencing accuracy.

[0060] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. An imaging agent that suppresses reduction in fluorescence intensity, characterized by, Consists of Tris, NaCl, ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid, sodium ascorbate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; wherein the concentration ratio of ethyl 3,4-dihydroxybenzoate, 3,4-dihydroxybenzoic acid and sodium ascorbate is 1:1:3, and pH=8.

0.

2. The imaging agent for suppressing reduction in fluorescence intensity according to claim 1, wherein The formulation consists of 90-110mM Tris, 750-850mM NaCl, 15-25mM ethyl 3,4-dihydroxybenzoate, 15-25mM 3,4-dihydroxybenzoic acid, 45-65mM sodium ascorbate, 0.1%-0.2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the rest is water, and pH=8.

0.

3. The imaging agent for suppressing reduction in fluorescence intensity according to claim 2, wherein The formulation consists of 100mM Tris, 800mM NaCl, 20mM ethyl 3,4-dihydroxybenzoate, 20mM 3,4-dihydroxybenzoic acid, 60mM sodium ascorbate, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the rest is water, and pH=8.

0.

4. A kit for nucleic acid sequencing, characterized by, The method comprises the following steps: extending the target nucleic acid molecule by the extension reagent; cleaving the reversible blocking moiety from the modified nucleotide molecule by the cleavage reagent; imaging the target nucleic acid molecule by the imaging reagent; and washing the target nucleic acid molecule by the washing reagent.

5. The kit for nucleic acid sequencing according to claim 4, wherein The modified nucleotide molecule is connected with a label molecule through a cleavable linker; the cleavable linker comprises one or more cleavable groups; the cleavable group comprises a disulfide bond and / or an azide; the cleavage reagent further comprises a component acting on the cleavable group to separate the label molecule from the modified nucleotide molecule; the reversible blocking moiety is a methyl azide; the washing reagent comprises an antioxidant and / or a cleavage reagent scavenger; and the extension reagent further comprises a polymerase.

6. The kit for nucleic acid sequencing according to claim 5, wherein The cleavage reagent comprises tri-(hydroxypropyl)phosphine.

7. The kit for nucleic acid sequencing according to any one of claims 4 to 6, wherein The washing reagent comprises cystine, thioctic acid, 3,3'-dithiodipropionic acid or polyethylene glycolated azide.

8. The kit for nucleic acid sequencing according to claim 7, wherein The washing reagent further comprises gallic acid and / or sodium ascorbate.

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