CN112601825B - Nucleic acid sequencing method - Google Patents

Nucleic acid sequencing method Download PDF

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CN112601825B
CN112601825B CN201880094670.0A CN201880094670A CN112601825B CN 112601825 B CN112601825 B CN 112601825B CN 201880094670 A CN201880094670 A CN 201880094670A CN 112601825 B CN112601825 B CN 112601825B
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nucleic acid
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CN112601825A (en
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刘二凯
陈奥
章文蔚
廖莎
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Qingdao Huada Zhizao Technology Co ltd
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Abstract

The invention provides a nucleic acid sequencing method and a corresponding sequencing device, wherein the method is characterized in that nucleotide analogues with fluorescent and phosphorescent groups are incorporated into the 3 'end of an initial growth nucleic acid chain when a duplex of a nucleic acid molecule to be detected is amplified, and the nucleotide at the 3' end of the nucleic acid molecule to be detected is judged by detecting fluorescent and phosphorescent signals.

Description

Nucleic acid sequencing method
Technical Field
The invention relates to the field of biological medicine, in particular to a nucleic acid sequencing method, nucleotide analogues, nucleotide analogue mixtures and application thereof.
Background
DNA sequencing techniques include the first generation DNA sequencing techniques represented by Sanger (Sanger) sequencing and the second generation DNA sequencing techniques represented by Illumina Hiseq2500, roche 454,ABI Solid,BGISEQ-500, etc. The Mulberry sequencing method has the characteristics of simple experimental operation, visual and accurate result, short experimental period and the like, and has wide application in the fields of clinical gene mutation detection, genotyping and the like with high requirement on timeliness of detection results. However, the disadvantages of the sanger sequencing method are small throughput and high cost, which limit its application in large-scale gene sequencing.
Compared with the first generation DNA sequencing technology, the second generation DNA sequencing technology has the characteristics of large sequencing flux, low cost, high automation degree and single molecule sequencing. Taking the Hiseq2500V2 sequencing technology as an example, one experimental flow can generate 10-200G base data, the average sequencing cost of each base is less than 1/1000 of the sequencing cost of the Sanger sequencing method, and the obtained sequencing result can be directly processed and analyzed by a computer. Thus, second generation DNA sequencing techniques are well suited for large scale sequencing.
The second generation of DNA sequencing technology developed today mainly involves sequencing-by-ligation (sequencing by ligation, SBL) and sequencing-by-synthesis (sequencing by synthesis, SBS) techniques. Typical examples of these sequencing techniques include SOLiD sequencing developed by Applied Biosystems, combined probe anchored ligation (cPAL) developed independently of Complete Genomics, combined probe anchored synthesis (cPAS) developed by Huada genes, illumina sequencing developed cooperatively by Illumina and Solexa technology, and the like. In these sequencing schemes, illumina and Complate Genomics employ methods of detecting optical signals, and in order to achieve identification and discrimination of 4 bases (A, T/U, C and G), it is generally necessary to use 4 fluorescent dyes to label the 4 bases separately. In this case, in order to read the fluorescent signals carried by the individual bases, the sequencing device must be equipped with at least 2 monochromatic excitation light sources and at least 2 cameras, which results in expensive manufacturing and huge volume of the sequencing device.
There have been studies reporting that discrimination and differentiation of 4 bases can be achieved by using 2 fluorescent dyes (Sara Goodwin, et al Nature Reviews Genetics 17,333-351 (2016)). For example, the NextSeq sequencing system and Mini-Seq sequencing system developed by Illumina corporation use a double fluorescent dye based sequencing method. In such sequencing methods, the identification and differentiation of 4 bases is achieved by different combinations of 2 fluorescent dyes. For example, four bases can be distinguished by labeling base A with a first fluorescent dye, base G with a second fluorescent dye, base C with both the first and second fluorescent dyes, and without labeling base T/U. In such sequencing methods, the sequencing device requires only one camera, but still needs to be equipped with at least 2 monochromatic excitation light sources. Thus, the manufacturing cost and volume of sequencing devices using 2 fluorescent dyes remains relatively high. In addition, the sequencing quality of the double-fluorescent dye-based sequencing method is significantly reduced compared to the sequencing method using 4 fluorescent dyes, mainly because of the difficulty in distinguishing the double-color fluorescence from the single-color fluorescence, and the accuracy is reduced. In the sequencing method using a fluorescent material, for example, illuminea iseq100, during the first and second signal acquisition, special reagents are added to the sequencing chip to realize signal conversion so as to distinguish different bases, thus the sequencing process is more complex and the duration is longer.
Thus, sequencing methods remain to be developed and improved.
Disclosure of Invention
In the prior art, the single fluorescence sequencing method mainly realizes signal conversion by using different chemical excision reactions and biotin/streptavidin interactions, and when the signal conversion is realized by using different chemical excision reactions and biotin/streptavidin interactions, a reaction reagent is added between the first image acquisition and the second image acquisition, so that the complexity of sequencing biochemistry is increased, the sequencing duration is prolonged, and the reagent cost is increased because certain signals are added by using small molecule and protein interactions. In the two-color fluorescence sequencing method in the prior art, two kinds of fluorescence are used for distinguishing four bases, and 2 kinds of lasers are required to excite fluorescent molecules with two different excitation wavelengths, so that the method is unfavorable for the miniaturization development of an instrument and cannot well reduce the cost of the instrument.
Based on the findings of the facts and problems, the inventor proposes a principle of distinguishing bases by using differences in optical properties of compounds for the first time, a sequencer based on the principle only needs one excitation light and a lens of a filtering device, and does not need to perform chemical reaction between photographing twice, so that hardware conditions and sequencing steps of the sequencer are simplified in principle, and the cost of the instrument and the cost of reagents are reduced. Meanwhile, the inventor also designs a new nucleotide analogue and designs a nucleotide analogue mixture, and the nucleotide analogue mixture can conveniently and accurately distinguish bases by utilizing the difference of optical properties of the nucleotide analogue mixture, so that DNA and/or RNA sequencing can be realized.
In a first aspect of the invention, the invention provides a method of sequencing a nucleic acid molecule. According to an embodiment of the invention, the method comprises: (1) Annealing a test nucleic acid molecule to a primer so as to form an initial duplex, wherein the test nucleic acid molecule or the primer is immobilized on a support in advance, the duplex is composed of the test nucleic acid molecule and the primer, and the duplex is immobilized on the support; (2) Incorporating one or both of the first through fifth nucleotide analogs into the 3 'end of the starting growing nucleic acid strand under the catalysis of a polymerase using the primer in the duplex as the first starting growing nucleic acid strand so as to extend only one first new nucleotide at the 3' end of the starting growing nucleic acid strand to form a first product duplex; (3) Removing the polymerase and unreacted first to fifth nucleotide analogs in the reaction systems of steps (1) and (2); (4) Judging a first nucleotide at the 3' -end of the nucleic acid molecule to be detected based on the fluorescent signal and the phosphorescent signal of the first product duplex; wherein the first to fifth nucleotide analogs have base complementary pairing ability, the hydroxyl group at the 3' position of ribose or deoxyribose of the first to fifth nucleotide analogs is protected by a protecting group that is a polymerase reaction blocking group, the first, second, third and fourth nucleotide analogs have different bases from the fifth nucleotide analog, the first and third nucleotide analogs have the same base as the third nucleotide analog, the first and second nucleotide analogs have different bases from the second nucleotide analog, the first and second nucleotide analogs have different bases from the fourth nucleotide analog, and the third nucleotide analog and the fourth nucleotide analog have different bases from the fourth nucleotide analog, the first and second nucleotide analogs each independently carry a fluorescent group, the fourth nucleotide analog carries a phosphorescent group, and the phosphorescent group. The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, and based on this principle, they devised a novel method of nucleic acid sequencing that uses the optical signals of the first to fifth nucleotide analogs described above (phosphorescence and fluorescence simultaneously, phosphorescence only without fluorescence, phosphorescence and fluorescence) to identify the corresponding bases in the nucleic acid molecule to be sequenced (A, T/U, C and G). With the sequencing method according to an embodiment of the present invention, only one excitation light and one filter device are required, and no compound reaction between the two detection signals is required. Therefore, the sequencing steps are simplified, the sequencing cost is reduced, and the sequencing result is accurate.
In a second aspect of the application, the application provides a nucleotide analogue. According to an embodiment of the present application, the nucleotide analog has a structural formula shown in formula (I),
wherein, base 1 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 1 Represents a phosphorescent group; c (C) 1 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 1 Represents a polymerase reaction blocking group; r is R 1 is-OH or-H, P 1 Is H or a phosphoric acid group. The inventors propose and design the nucleotide analogues carrying phosphorescent groups for the first time, the nucleotide analogues carrying phosphorescent groups can be independently used in DNA and/or RNA sequencing, the nucleotide analogues carrying fluorescent groups in the sequencing methods used for fluorescent dyes in the prior art such as a bicolor fluorescent sequencing method and a monochromatic fluorescent sequencing method are replaced, and meanwhile, the sequencing method for distinguishing bases by utilizing the optical property difference of the nucleotide analogues carrying phosphorescent groups and the optical property difference of the nucleotide analogues can be realized. The nucleotide analogue according to the embodiment of the application carries a phosphorescent group and is a novel nucleotide analogue which can be used in DNA and/or RNA sequencing.
In a third aspect of the invention, the invention provides a mixture of nucleotide analogs. According to an embodiment of the invention, the nucleotide analogue mixture comprises the nucleotide analogues described previously. The inventors propose for the first time a nucleotide analogue mixture comprising nucleotide analogues carrying phosphorescent groups, whereby the nucleotide analogues carrying phosphorescent groups in the nucleotide analogue mixture can be used to generate phosphorescent optical signals for sequencing of nucleic acids.
In a fourth aspect of the invention, the invention provides a mixture of nucleotide analogs. According to an embodiment of the invention, the nucleotide analogue mixture comprises: a first nucleotide analogue and a second nucleotide analogue, each independently having a structural formula shown in formula II,
wherein, base 2 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 2 Represents a fluorescent group; c (C) 2 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 2 Represents a polymerase reaction blocking group; r is R 2 is-OH or-H; p (P) 2 Represents H or a phosphate group;
a third nucleotide analogue and a fourth nucleotide analogue, each independently having a structural formula shown in formula I,
Wherein, base 1 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 1 Represents a phosphorescent group; c (C) 1 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 1 Represents a polymerase reaction blocking group; r is R 1 is-OH or-H; p (P) 1 Is H or a phosphate group;
a fifth nucleotide analog having a structural formula shown in formula III,
wherein, base 3 Represents adenine, guanine, cytosine thymine or uracil; b (B) 3 Representing aggregationAn enzyme reaction blocking group; r is R 3 is-OH or-H; p (P) 3 Is H or a phosphate group;
wherein the first, second, third and fourth nucleotide analogs have different bases than the fifth nucleotide analog; the first nucleotide analogue has the same base as the third nucleotide analogue; the first nucleotide analog and the second nucleotide analog have different bases; the first nucleotide analogue and the second nucleotide analogue have different bases from the fourth nucleotide analogue; and the third nucleotide analogue and the fourth nucleotide analogue have different bases. The inventors have proposed for the first time that the difference in optical properties of compounds is used to distinguish bases, and based on this principle, they devised a nucleotide analog mixture, and by using the above-mentioned first to fifth nucleotide analogs for nucleic acid sequencing, the type of the corresponding base on the nucleic acid strand can be rapidly and accurately identified by detecting different optical signals (phosphorescence and fluorescence at the same time, phosphorescence and fluorescence only, phosphorescence and fluorescence only), sequencing is performed using the nucleotide analog mixture according to the embodiment of the present invention, only one excitation light and one filter device are needed, and no compound reaction is needed between the two detection signals, thereby simplifying the sequencing step, reducing the sequencing cost, and the sequencing result is accurate.
In a fifth aspect of the application, the application provides a kit. According to an embodiment of the application, the kit comprises: a nucleotide analogue as described hereinbefore, or a mixture of nucleotide analogues as described hereinbefore. The inventors propose and design a nucleotide analogue carrying a phosphorescent group for the first time, and the nucleotide analogue carrying the phosphorescent group can be independently used in DNA and/or RNA sequencing to replace the nucleotide analogue carrying the fluorescent group in the sequencing method using fluorescent dye in the two-color fluorescence sequencing method, the single-color fluorescence sequencing method and the like in the prior art. Meanwhile, by utilizing the difference of optical properties of the nucleotide analogues carrying phosphorescent groups and the nucleotide analogues carrying fluorescent groups, the sequencing method for distinguishing bases by utilizing the difference of the optical properties of the compounds, which is proposed for the first time, can be realized, so that the nucleotide analogue mixture is designed, and the nucleotide analogue mixture is applied to the preparation of a kit. The kit provided by the embodiment of the application has the advantages of low cost and simple and convenient operation, and can rapidly and accurately detect the type of the base in the nucleic acid molecule.
In a sixth aspect of the application, the application provides a method of performing a polymerase reaction. According to an embodiment of the application, the method comprises: (1) The mixture comprising the single stranded template, the primer, the mixture of nucleotide analogs described above, and the polymerase is placed under conditions suitable for primer extension, wherein the primer matches a portion of the single stranded template such that only one first new nucleotide extends at the 3' end of the primer. The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, and based on this principle, they devised a mixture of nucleotide analogs, using the first to fifth nucleotide analogs described above for nucleic acid sequencing, to accurately identify the type of corresponding base in a nucleic acid strand by detecting different optical signals (phosphorescence and fluorescence simultaneously, phosphorescence and fluorescence only, phosphorescence and phosphorescence only, phosphorescence and fluorescence only). According to the method provided by the embodiment of the application, the nucleotide analogue can be precisely paired with the nucleic acid chain, so that the nucleic acid sequencing can be conveniently, rapidly and accurately performed.
In a seventh aspect of the invention, the invention provides a method of nucleic acid sequencing. According to an embodiment of the invention, the method comprises: the nucleic acid sequence to be sequenced is subjected to a controlled chain polymerase reaction using the methods described previously. According to the method provided by the embodiment of the invention, only one excitation light and one lens of the filtering device are needed in the sequencing process, and chemical reaction between two photographing is not needed, so that the sequencing step is simplified, the sequencing time length is reduced, the sequencing cost is reduced, and the sequencing result is accurate.
In an eighth aspect of the invention, the invention provides a sequencer. According to an embodiment of the present invention, the sequencer includes: a housing; a chain polymerase reaction region disposed in the housing; an excitation light emitter adapted to emit excitation light of a predetermined wavelength toward the chain polymerase reaction region; and a signal acquisition device adapted to acquire fluorescent and phosphorescent signals in the chain polymerase reaction region. The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, based on this principle, they devised a nucleotide analogue mixture, using the above first to fifth nucleotide analogues for nucleic acid sequencing, by detecting different optical signals (phosphorescence and fluorescence simultaneously, phosphorescence and fluorescence only) to identify the type of base corresponding to a nucleic acid strand, and based on the above sequencing method, they proposed a novel sequencer which required only one excitation light and one lens of a filter device, and did not require a chemical reaction between two shots, simplifying the hardware conditions and sequencing steps of the sequencer, and reducing the instrument cost and reagent cost. The sequencer provided by the embodiment of the invention has the advantages of low cost, simple sequencing process, short sequencing time and accurate sequencing result, and is beneficial to the miniaturization development of the sequencer.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a sequencer according to an embodiment of the present invention;
FIG. 2 is a sequencer (including a controller) according to an embodiment of the present invention;
fig. 3 is an optical channel image according to an embodiment of the present invention, wherein the left side is a fluorescent channel image and the right side is a phosphorescent channel image, wherein reference numerals: sequencer 1000, casing 100, chain polymerase reaction region 11, laser emitter 12, signal acquisition device 13, controller 14.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
Method for sequencing nucleic acid molecules
In a first aspect of the invention, the invention provides a method of sequencing a nucleic acid molecule. According to an embodiment of the invention, the method comprises: (1) Annealing a test nucleic acid molecule to a primer so as to form an initial duplex, wherein the test nucleic acid molecule or the primer is immobilized on a support in advance, the duplex is composed of the test nucleic acid molecule and the primer, and the duplex is immobilized on the support; (2) Incorporating one or both of the first through fifth nucleotide analogs into the 3 'end of the starting growing nucleic acid strand under the catalysis of a polymerase using the primer in the duplex as the first starting growing nucleic acid strand so as to extend only one first new nucleotide at the 3' end of the starting growing nucleic acid strand to form a first product duplex; (3) Removing the polymerase and unreacted first to fifth nucleotide analogs in the reaction systems of steps (1) and (2); (4) Judging a first nucleotide at the 3' -end of the nucleic acid molecule to be detected based on the fluorescent signal and the phosphorescent signal of the first product duplex; wherein the first to fifth nucleotide analogs have base complementary pairing ability, the hydroxyl group at the 3' position of ribose or deoxyribose of the first to fifth nucleotide analogs is protected by a protecting group that is a polymerase reaction blocking group, the first, second, third and fourth nucleotide analogs have different bases from the fifth nucleotide analog, the first and third nucleotide analogs have the same base as the third nucleotide analog, the first and second nucleotide analogs have different bases from the second nucleotide analog, the first and second nucleotide analogs have different bases from the fourth nucleotide analog, the third nucleotide analog has different bases from the fourth nucleotide analog, the first and second nucleotide analogs each independently carry a fluorescent group, the fourth nucleotide analog does not carry a fluorescent group, and the phosphorescent group. The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, and based on this principle, they devised a novel method of nucleic acid sequencing that uses the optical signals of the first to fifth nucleotide analogs described above (phosphorescence and fluorescence simultaneously, phosphorescence only without fluorescence, phosphorescence and fluorescence) to identify the corresponding bases in the nucleic acid molecule to be sequenced (A, T/U, C and G). With the sequencing method according to an embodiment of the present invention, only one excitation light and one filter device are required, and no compound reaction between the two detection signals is required. Therefore, the sequencing steps are simplified, the sequencing cost is reduced, and the sequencing result is accurate.
According to an embodiment of the invention, the method further comprises: (5) Subjecting the first product duplex to cleavage treatment in a reaction system comprising a solution phase and a solid phase to remove protecting groups and/or optical signal groups at the 3' position of ribose or deoxyribose in the nucleotide analog, (6) removing the solution phase of the reaction system in step (5), (7) incorporating one or both of the first through fifth nucleotide analogs into the 3' end of the starting growing nucleic acid strand under the catalysis of a polymerase with the cleavage treatment product in the reaction system of step (6) as a second initially growing nucleic acid strand to extend only one second new nucleotide at the 3' end of the second initially growing nucleic acid strand to form a second product duplex; (8) Repeating the steps (3) and (4), and judging the second nucleotide sequence of the 3' -end of the nucleic acid molecule to be detected. By cleaving the protecting group at the 3' position on the nucleotide analog in order to ensure the next round of the polymer chain reaction, and by cleaving the luminescent group (fluorescent group or phosphorescent group) on the nucleotide analog in order to better detect the optical signal carried by the nucleotide analog on the next round of the polymerase chain reaction amplification. In the present invention, the solution phase means a reaction solution in which a reaction occurs, and the solid phase means a support to which a template is immobilized.
According to an embodiment of the invention, the extended first new nucleotide is a first or second nucleotide analogue and the cleaved light signaling group is a fluorescent group.
According to an embodiment of the invention, the extended first new nucleotide is a third, fourth nucleotide analogue and the cleaved optical signal group is a phosphorescent group.
According to an embodiment of the invention, the extended first new nucleotide is a fifth nucleotide analogue, cleaving only the protecting group at the 3' position of ribose or deoxyribose from the protecting groups.
According to an embodiment of the invention, the method further comprises the following step (9): the method further comprises the following step (9): (9) Repeating steps (5) - (8) one or more times to determine the nucleotide sequence of the nucleic acid molecule to be tested. Repeating steps (5) - (8) one or more times until the complete segment of the nucleic acid molecule to be sequenced is sequenced.
According to an embodiment of the present invention, the presence of both the fluorescent signal and the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the first nucleotide analogue and the third nucleotide analogue, the absence of both the fluorescent signal and the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the fifth nucleotide analogue, the presence of the fluorescent signal but the absence of the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the second nucleotide analogue, the absence of the fluorescent signal but the presence of the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the fourth nucleotide analogue. Thus, the bases corresponding to the nucleic acid molecules to be sequenced can be determined rapidly and accurately by different optical signals.
According to an embodiment of the application, the nucleic acid molecule to be detected is DNA, which has been subjected to a denaturation treatment in advance so as to obtain a single-stranded nucleic acid molecule to be detected.
Nucleotide analogues
In a second aspect of the application, the application provides a nucleotide analogue. According to an embodiment of the present application, the nucleotide analog has a structural formula shown in formula (I),
wherein, base 1 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 1 Represents a phosphorescent group; c (C) 1 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 1 Represents a polymerase reaction blocking group; r is R 1 is-OH or-H, P 1 Is H or a phosphoric acid group. The inventors propose and design the nucleotide analogues carrying phosphorescent groups for the first time, the nucleotide analogues carrying phosphorescent groups can be independently used in DNA and/or RNA sequencing, the nucleotide analogues carrying fluorescent groups in the sequencing methods used for fluorescent dyes in the prior art such as a bicolor fluorescent sequencing method and a monochromatic fluorescent sequencing method are replaced, and meanwhile, the sequencing method for distinguishing bases by utilizing the optical property difference of the nucleotide analogues carrying phosphorescent groups and the optical property difference of the nucleotide analogues can be realized. The nucleotide analogue according to the embodiment of the application carries a phosphorescent group and is a novel nucleotide analogue which can be used in DNA and/or RNA sequencing.
The phosphorescent group is not particularly limited as long as the phosphorescent group satisfies a phosphorescent signal capable of controllably detecting emission under a specific excitation light condition. For example, the phosphorescent group may have the following structure:
wherein the R is 4 H, F, cl, br, I, CN, NO of a shape of H, F, cl, br, I, CN, NO 2 、C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 An alkoxy group. The above groups can phosphoresce under the action of specific excitation light.
According to an embodiment of the invention, the phosphate groups are mono-, di-, tri-or poly-phosphate groups.
The cleavable group is not particularly limited as long as it is cleaved under specific conditions. For example, the cleavable group may be a group comprising-S-, CH 2 CH＝CH 2 、-CH 2 N 3 And the like, and the cleavable groups commonly found in the prior art.
According to an embodiment of the invention, the C 1 Further comprising a polymerase reaction blocking group. It should be noted that if the cleavable group includes a polymerase reaction site, a polymerase reaction blocking group needs to be introduced into the polymerase reaction site to prevent the polymerase chain reaction or the reaction at the site during the sequencing from affecting the sequencing result.
For example, the C 1 Including but not limited to the following structures:
wherein n1 to n17 are each independently an integer of 0 to 7.
The polymerase reaction blocking group is not particularly limited as long as it can block the site to perform a polymerase reaction under a specific condition and can be removed to perform a polymerase reaction under another specific condition. For example, the polymerase reaction blocking group may be a group comprising-S-, CH 2 CH＝CH 2 、-CH 2 N 3 And the like, and are common in the prior art. The above groups allow for controlled amplification of only one new nucleotide at a time during the polymerase chain reaction or sequencing process, after which the optical signal is detected and then excised for the next round of amplification.
According to an embodiment of the present invention, the structure of formula (I) is one of the following:
nucleotide analogue mixture
In a third aspect of the invention, the invention provides a mixture of nucleotide analogs. According to an embodiment of the invention, the nucleotide analogue mixture comprises the nucleotide analogues described previously. The inventors propose for the first time a nucleotide analogue mixture comprising nucleotide analogues carrying phosphorescent groups, whereby the nucleotide analogues carrying phosphorescent groups in the nucleotide analogue mixture can be used to generate phosphorescent optical signals for sequencing of nucleic acids.
Nucleotide analogue mixture
In a fourth aspect of the invention, the invention provides a mixture of nucleotide analogs. According to an embodiment of the invention, the nucleotide analogue mixture comprises: a first nucleotide analogue and a second nucleotide analogue, each independently having a structural formula shown in formula II,
wherein, base 2 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 2 Represents a fluorescent group; c (C) 2 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 2 Represents a polymerase reaction blocking group; r is R 2 is-OH or-H; p (P) 2 Represents H or a phosphate group;
a third nucleotide analogue and a fourth nucleotide analogue, each independently having a structural formula shown in formula I,
wherein, base 1 Represents adenine, guanine, cytosine, thymine or uracil; d (D) 1 Represents a phosphorescent group; c (C) 1 Represents a cleavable bond or an optionally substituted cleavable group; b (B) 1 Represents a polymerase reaction blocking group; r is R 1 is-OH or-H; p (P) 1 Is H or a phosphate group;
a fifth nucleotide analog having a structural formula shown in formula III,
wherein, base 3 Represents adenine, guanine, cytosine thymine or uracil; b (B) 3 Represents a polymerase reaction blocking group; r is R 3 is-OH or-H; p (P) 3 Is H or a phosphate group;
wherein the first, second, third and fourth nucleotide analogs have different bases than the fifth nucleotide analog; the first nucleotide analogue has the same base as the third nucleotide analogue; the first nucleotide analog and the second nucleotide analog have different bases; the first nucleotide analogue and the second nucleotide analogue have different bases from the fourth nucleotide analogue; and the third nucleotide analogue and the fourth nucleotide analogue have different bases. The inventors have proposed for the first time that the difference in optical properties of compounds is used to distinguish bases, and based on this principle, they devised a nucleotide analog mixture, and by using the above-mentioned first to fifth nucleotide analogs for nucleic acid sequencing, the type of the corresponding base on the nucleic acid strand can be rapidly and accurately identified by detecting different optical signals (phosphorescence and fluorescence at the same time, phosphorescence and fluorescence only, phosphorescence and fluorescence only), sequencing is performed using the nucleotide analog mixture according to the embodiment of the present invention, only one excitation light and one filter device are needed, and no compound reaction is needed between the two detection signals, thereby simplifying the sequencing step, reducing the sequencing cost, and the sequencing result is accurate.
The phosphorescent group is not particularly limited as long as the phosphorescent group satisfies a phosphorescent signal capable of controllably detecting emission under a specific excitation light condition. For example, the phosphorescent group may have the following structure:
wherein the R is 4 H, F, cl, br, I, CN, NO of a shape of H, F, cl, br, I, CN, NO 2 、C 1-6 Alkyl, C 1-6 Haloalkyl, C 1-6 An alkoxy group. The above groups can phosphoresce under the action of specific excitation light.
According to an embodiment of the invention, the phosphate groups are mono-, di-, tri-or poly-phosphate groups.
The cleavable group is not particularly limited as long as it is cleaved under specific conditions. For example, the cleavable group may be a group comprising-S-, CH 2 CH＝CH 2 、-CH 2 N 3 And the like, and the cleavable groups commonly found in the prior art.
According to an embodiment of the invention, the C 1 And/or C 2 Further comprising a polymerase reaction blocking group. It should be noted that if the cleavable group includes a polymerase reaction site, a polymerase reaction blocking group needs to be introduced into the polymerase reaction site to prevent the polymerase chain reaction or the reaction at the site during the sequencing from affecting the sequencing result.
For example, the C 1 And/or C 2 Including but not limited to the following structures:
wherein n1 to n17 are each independently an integer of 0 to 7.
The polymerase reaction blocking group is not particularly limited, and may be any oneIt is sufficient to block the site for the polymerase reaction under specific conditions and to remove it for the polymerase reaction under another specific condition. For example, the polymerase reaction blocking group may be a group comprising-S-, CH 2 CH＝CH 2 、-CH 2 N 3 And the like, and are common in the prior art. The above groups allow for controlled amplification of only one new nucleotide at a time during the polymerase chain reaction or sequencing process, after which the optical signal is detected and then excised for the next round of amplification.
The fluorescent group is not particularly limited as long as the fluorescent group can emit a controllably detectable fluorescent signal under the condition of specific excitation light. For example D 2 Including but not limited to the following structures:
according to an embodiment of the present invention, the structure of formula (I) is one of the following:
/>
according to one embodiment of the invention, the first nucleotide analogue has a structure represented by formula (5):
the second nucleotide analogue has a structure represented by formula (6):
The third nucleotide analogue has a structure represented by formula (2):
the fourth nucleotide analogue has a structure represented by formula (1):
the fifth nucleotide analog has a structure represented by formula (7):
the nucleotide analog mixture having the above structure is only one of the nucleotide analog mixtures that can be used to implement the sequencing method described above.
Kit for detecting a substance in a sample
In a fifth aspect of the application, the application provides a kit. According to an embodiment of the application, the kit comprises: a nucleotide analogue as described hereinbefore, or a mixture of nucleotide analogues as described hereinbefore. The inventors propose and design a nucleotide analogue carrying a phosphorescent group for the first time, and the nucleotide analogue carrying the phosphorescent group can be independently used in DNA and/or RNA sequencing to replace the nucleotide analogue carrying the fluorescent group in the sequencing method using fluorescent dye in the two-color fluorescence sequencing method, the single-color fluorescence sequencing method and the like in the prior art. Meanwhile, by utilizing the difference of optical properties of the nucleotide analogues carrying phosphorescent groups and the nucleotide analogues carrying fluorescent groups, the sequencing method for distinguishing bases by utilizing the difference of the optical properties of the compounds, which is proposed for the first time, can be realized, so that the nucleotide analogue mixture is designed, and the nucleotide analogue mixture is applied to the preparation of a kit. The kit provided by the embodiment of the application has the advantages of low cost and simple and convenient operation, and can rapidly and accurately detect the type of the base in the nucleic acid molecule.
According to an embodiment of the invention, the kit further comprises: a cleaving reagent that can act on a cleavable group or a cleavable bond. The cleavable group or bond in the aforementioned nucleotide analogue or nucleotide analogue mixture can be cleaved using the cleavage reagent described above, thereby allowing removal of the polymerase reaction blocking group and exposing the site of the polymerase reaction for the next new base amplification.
The cleavage reagent is not particularly limited as long as it is capable of cleaving a cleavable bond or a cleavable group in the nucleotide analog and does not substantially affect the nucleic acid molecule to be sequenced and the polymerase chain reaction. For example, TCEP/THPP can be used as a cleavage reagent to cleave disulfide bonds with high efficiency, and organic phosphonates can be used as cleavage reagents to cleave azide groups with high efficiency.
Method of polymerase reaction
In a sixth aspect of the invention, the invention provides a method of performing a polymerase reaction. According to an embodiment of the invention, the method comprises: (1) The mixture comprising the single stranded template, the primer, the mixture of nucleotide analogs described above, and the polymerase is placed under conditions suitable for primer extension, wherein the primer matches a portion of the single stranded template such that only one first new nucleotide extends at the 3' end of the primer. The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, and based on this principle, they devised a mixture of nucleotide analogs, using the first to fifth nucleotide analogs described above for nucleic acid sequencing, to accurately identify the type of corresponding base in a nucleic acid strand by detecting different optical signals (phosphorescence and fluorescence simultaneously, phosphorescence and fluorescence only, phosphorescence and phosphorescence only, phosphorescence and fluorescence only). According to the method provided by the embodiment of the invention, the nucleotide analogue can be precisely paired with the nucleic acid chain, so that the nucleic acid sequencing can be conveniently, rapidly and accurately performed.
According to an embodiment of the invention, the single stranded template or primer is immobilized on a solid support.
According to an embodiment of the invention, the single stranded template or primer is immobilized on a chip.
According to an embodiment of the invention, the method further comprises: (2) Cleaving the polymerase reaction blocking group of the new nucleotide and returning to step (1) to continue extension of only one second new base at the 3' end of the first new nucleotide. The method is beneficial to accurately and rapidly sequencing nucleic acid molecules.
According to an embodiment of the present invention, after step (1), further comprising: (1-1) detecting a fluorescent signal and a phosphorescent signal of the first new base, respectively. By detecting the optical signal of the first new base, the type of the first new base can be accurately identified.
According to an embodiment of the present invention, before step (1-1), further comprising: removing the reactants in the system after extending the first new nucleotide. By removing the reactant in the system after extending the first new nucleotide, and then performing optical signal detection, the sensitivity and accuracy of the optical signal detection can be improved.
According to an embodiment of the present invention, after step (1-1), further comprising: (1-2) determining the type of at least one of the first new base and the base at the position on the single-stranded template corresponding to the first new base based on at least one of the fluorescent signal and the phosphorescent signal.
According to an embodiment of the present invention, the fluorescent signal and the phosphorescent signal are present simultaneously with the base indicating the first new nucleotide being the base corresponding to the first nucleotide analogue and the third nucleotide analogue, and neither the fluorescent signal nor the phosphorescent signal is present with the base indicating the first new nucleotide being the base corresponding to the fifth nucleotide analogue, and the fluorescent signal is present but the phosphorescent signal is not present with the base indicating the first new nucleotide being the base corresponding to the second nucleotide analogue, and the fluorescent signal is not present but the phosphorescent signal is present but the base indicating the first new nucleotide is the base corresponding to the fourth nucleotide analogue. Thus, the base type of the corresponding position in the nucleic acid molecule to be sequenced can be accurately identified according to the difference of the optical signals of the bases of the first new nucleotide.
According to an embodiment of the present invention, in step (1-2), an excitation wavelength of 400 to 480nm and a signal acquisition filter of 500 to 570nm are employed. The inventor finds that on the basis of the excitation wavelength and the signal acquisition filter, the fluorescent signal and the phosphorescent signal can be detected successively, and the detection requirement can be met only by one excitation light and one signal acquisition filter, and meanwhile, chemical reaction is not needed in the two signal acquisition processes. Thus, sequencing time and sequencing cost are saved.
According to an embodiment of the invention, the fluorescence signal is collected during a period when the excitation light is on, and the phosphorescence signal is collected between 0.5 and 100 milliseconds after the excitation light is off. The method provided by the embodiment of the invention is used for carrying out the polymerase reaction, chemical reaction is not needed between two signal acquisitions, and the time interval between the two signal acquisitions is short. Thus, sequencing time and sequencing cost are saved.
Method for determining nucleic acid sequence
In a seventh aspect of the invention, the invention provides a method of nucleic acid sequencing. According to an embodiment of the invention, the method comprises: the nucleic acid sequence to be sequenced is subjected to a controlled chain polymerase reaction using the methods described previously. According to the method provided by the embodiment of the invention, only one excitation light and one lens of the filtering device are needed in the sequencing process, and chemical reaction between two photographing is not needed, so that the sequencing step is simplified, the sequencing time length is reduced, the sequencing cost is reduced, and the sequencing result is accurate.
Sequencer
In a sixth aspect of the invention, the invention provides a sequencer. Referring to fig. 1, according to an embodiment of the present invention, the sequencer 1000 includes: a housing 100;
A chain polymerase reaction region 11, the chain polymerase reaction region 11 being disposed in the housing 100;
an excitation light emitter 12, said laser light emitter 12 being adapted to emit excitation light of a predetermined wavelength towards said chain polymerase reaction region 11; according to a specific embodiment of the present invention, the predetermined wavelength is 400 to 480nm.
And a signal acquisition device 13, said signal acquisition device 13 being adapted to acquire fluorescent and phosphorescent signals in said chain polymerase reaction region 11.
According to yet another embodiment of the present invention, referring to fig. 2, the sequencer 1000 further comprises a controller 14, wherein the controller 14 is respectively connected to the signal acquisition device 13 and the laser emitter 12, for controlling the on and off of the laser emitter 12, and controlling the signal acquisition device 13 to switch between acquiring fluorescent signals and phosphorescent signals. According to a further embodiment of the invention, the controller 14 is adapted to control the signal acquisition means 13 to acquire a fluorescent signal during activation of the excitation light emitters 12, the phosphorescent signal being acquired within a predetermined time frame after the excitation light emitters 12 are turned off. According to yet another embodiment of the invention, the predetermined time is 0.5 to 100 milliseconds.
The inventors have proposed for the first time the use of differences in the optical properties of compounds to distinguish bases, based on this principle, they devised a nucleotide analogue mixture, using the above first to fifth nucleotide analogues for nucleic acid sequencing, by detecting different optical signals (phosphorescence and fluorescence simultaneously, phosphorescence and fluorescence only) to identify the type of base corresponding to a nucleic acid strand, and based on the above sequencing method, they proposed a novel sequencer which required only one excitation light and one lens of a filter device, and did not require a chemical reaction between two shots, simplifying the hardware conditions and sequencing steps of the sequencer, and reducing the instrument cost and reagent cost. The sequencer provided by the embodiment of the application has the advantages of low cost, simple sequencing process, short sequencing time and accurate sequencing result, and is beneficial to the miniaturization development of the sequencer.
Preparation method
The compounds represented by the formula (II) and the formula (III) in the nucleotide analogue mixture related to the application are common nucleotide analogues in the prior art, can be synthesized by referring to methods in the prior art, for example, can be synthesized by referring to methods in patents such as WO 2017/058953 A1, WO 2017/087887A1 and US 2017/0130551 A1.
The compound shown in the formula (I) in the nucleotide analogue mixture is a nucleotide analogue which is first proposed by the inventor, and the synthesis method can also be synthesized by referring to the method in the prior art, except that the compound is synthesized by converting a fluorescent group into a phosphorescent group in the synthesis process. The synthesis of phosphorescent groups can be carried out with reference to the methods of synthesis of the relevant phosphorescent groups of the prior art, for example with reference to the methods in Fraser, C.L., NATURE MATERIALS,2009,08,747-751 (DOI: 10.1038/NMAT 2509).
For example, using Fraser, c.l., the procedure in the literature of natu MATERIALS,2009,08,747-751 (DOI: 10.1038/NMAT 2509) is available as starting material 1:
n may be any integer (including 0) as desired,
raw material 2 with a fluorescent group to be linked (raw material 2 to be regarded as a phosphorescent group to be linked in the present application) is obtained by the method in WO 2017/058953 A1, WO 2017/087887A1, US 2017/0130551 A1, etc.;
then the above-mentioned raw material 1 and raw material 2 to be connected with phosphorescent group are undergone the processes of classical chemical reaction of condensation reaction, substitution reaction or addition reaction, etc. under the proper condition to make synthesis so as to obtain the nucleotide analogue with phosphorescent group.
Example 1:
the inventors validated the sequencing method by using the following compounds:
the optical signals carried by the above compounds are shown in table 1:
table 1: optical signal information
Base type Fluorescent channel Phosphorescent light channel
A Luminous device Non-luminous
G Non-luminous Non-luminous
C Non-luminous Luminous device
T Luminous device Luminous device
Wherein, the T base uses a mixture of phosphorescence modified dTTP and fluorescence modified dTTP.
In order to conduct the base discrimination verification based on this principle, a plurality of DNA of different sequences were immobilized on a glass chip by a spotter under a fluorescence microscope, respectively. The diameter of each point is 50 micrometers, the excitation wavelength of the fluorescence microscope is selected to be 400-480 nanometers, and the range of a fluorescence and phosphorescence acquisition signal filter is 500-570 nanometers. Fluorescent signal acquisition is during the on period of excitation light and phosphorescent signal acquisition is between 0.5ms and 100ms after excitation light is off.
In the verification experiment, the enzyme in the BGISEQ-500 reagent and various buffers are adopted as the reagent. Wherein the fluorescence modified probes of the sequencing reagent are changed into the 5 fluorescence and phosphorescence modified reversible blocking nucleotides.
First, a DNA array (Boao biochip custom service) on a silicon wafer was purchased, four DNA sequences were used, and four base sequences were contained in each position corresponding to the region to be tested, wherein the four base sequences are shown in Table 2.
Table 2: sequence information
After the DNA was immobilized, sequencing primers for partial complementary sequences were added (table 2 above). After adding a DNA polymerase mixture containing the above five nucleotides to a silicon wafer, the 3' end of each DNA primer is polymerized with a corresponding base under PCR extension conditions, unreacted nucleotides are washed off, phosphate buffer containing vitamin C is added, image acquisition is performed under a fluorescence microscope, and a phosphorescent signal image can be obtained by setting the acquisition signal time of the microscope.
The first cycle first obtains the image of the fluorescent channel on the left and the phosphorescent channel on the right, wherein the leftmost two columns are mixed DNA localization areas, so that 6 points have signals in both channels, and 3-10 columns are single-kind DNA sequence points, and the total number of the points is 6 multiplied by 8.
Sequencing through 5 cycles respectively judges the sequence of the 48 points as follows;
4a, 4e, 4f, 5a, 5e, 6a, 6c, 7a, 8b, 8c, 8f, 9b, 9c, 9e, 10c, 10d and 10e are sequence 1.
3b, 3c, 3e, 4c, 6b, 7b, 9a and 10b are sequence 2.
3a, 4d, 5b, 5c, 6f, 7d, 7f, 8a, 9f and 10a are sequence 3.
3d, 3f, 4b, 5d, 5f, 6d, 6e, 7c, 7e, 8d, 8e, 9d and 10f are sequence 4.
The sequence is identical to the sequence designed by the custom chip, and 100% accuracy is achieved in short sequencing read length.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (23)

1. A method for sequencing a nucleic acid molecule, characterized in that,
(1) Annealing a test nucleic acid molecule to a primer so as to form an initial duplex, wherein the test nucleic acid molecule or the primer is immobilized on a support in advance, the duplex is composed of the test nucleic acid molecule and the primer, and the duplex is immobilized on the support;
(2) Incorporating one or both of the first through fifth nucleotide analogs into the 3 'end of the starting growing nucleic acid strand under the catalysis of a polymerase using the primer in the duplex as the first starting growing nucleic acid strand so as to extend only one first new nucleotide at the 3' end of the starting growing nucleic acid strand to form a first product duplex;
(3) Removing the polymerase and unreacted first to fifth nucleotide analogs in the reaction systems of steps (1) and (2);
(4) Judging a first nucleotide at the 3' -end of the nucleic acid molecule to be detected based on the fluorescent signal and the phosphorescent signal of the first product duplex;
wherein the first to fifth nucleotide analogs have base complementary pairing ability, the hydroxyl group at the 3' -position of ribose or deoxyribose of the first to fifth nucleotide analogs is protected by a protecting group, the protecting group is a polymerase reaction blocking group,
The first, second, third and fourth nucleotide analogs have different bases than the fifth nucleotide analog,
the first nucleotide analogue has the same base as the third nucleotide analogue,
the first nucleotide analogue and the second nucleotide analogue have different bases,
the first nucleotide analogue and the second nucleotide analogue have different bases from the fourth nucleotide analogue,
the third nucleotide analogue and the fourth nucleotide analogue have different bases,
the first and second nucleotide analogs each independently carrying a fluorescent group and not carrying a phosphorescent group,
the third and fourth nucleotide analogs each independently carrying a phosphorescent moiety and not carrying a fluorescent moiety,
the fifth nucleotide analog does not carry a fluorescent group and a phosphorescent group.
2. The method as recited in claim 1, further comprising:
(5) Subjecting the first product duplex to cleavage treatment in a reaction system comprising a solution phase and a solid phase in order to remove protecting groups and/or optical signal groups at the 3' position of ribose or deoxyribose in the nucleotide analog,
(6) Removing the solution phase of the reaction system in the step (5),
(7) Taking the cleavage treatment product in the reaction system of the step (6) as a second initial growth nucleic acid strand, and incorporating one or two of the first to fifth nucleotide analogs into the 3 'end of the initial growth nucleic acid strand under the catalysis of polymerase so as to extend only one second new nucleotide at the 3' end of the second initial growth nucleic acid strand to form a second product duplex;
(8) Repeating the steps (3) and (4), and judging the second nucleotide sequence of the 3' -end of the nucleic acid molecule to be detected.
3. The method of claim 2, wherein the extended first new nucleotide is a first or second nucleotide analogue and the cleaved optical signal group is a fluorescent group;
optionally, the extended first new nucleotide is a third, fourth nucleotide analogue, and the cleaved light signaling group is a phosphorescent group;
optionally, the extended first new nucleotide is a fifth nucleotide analogue that cleaves only the protecting group at the 3' position of ribose or deoxyribose in the protecting group.
4. The method according to claim 2, characterized in that the method further comprises the following step (9):
(9) Repeating steps (5) - (8) one or more times to determine the nucleotide sequence of the nucleic acid molecule to be tested.
5. The method according to any one of claims 1 to 4, wherein the presence of both the fluorescent signal and the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the first nucleotide analogue and the third nucleotide analogue,
the absence of both the fluorescent signal and the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the fifth nucleotide analog,
the presence of the fluorescent signal, but the absence of the phosphorescent signal, indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the second nucleotide analog,
the presence of the fluorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the base corresponding to the fourth nucleotide analog;
optionally, the nucleic acid molecule to be detected is DNA, and the nucleic acid molecule to be detected is subjected to denaturation treatment in advance so as to obtain a single-stranded nucleic acid molecule to be detected.
6. A mixture of nucleotide analogs comprising:
a first nucleotide analogue and a second nucleotide analogue, each independently having a structural formula shown in formula II,
Wherein,
Base 2 represents adenine, guanine, cytosine, thymine or uracil;
D 2 represents a fluorescent group;
C 2 represents a cleavable bond or an optionally substituted cleavable group;
B 2 represents a polymerase reaction blocking group;
R 2 is-OH or-H;
P 2 represents H or a phosphate group;
a third nucleotide analogue and a fourth nucleotide analogue, each independently having a structural formula shown in formula I,
wherein,
Base 1 represents adenine, guanine, cytosine, thymine or uracil;
D 1 represents a phosphorescent group;
C 1 represents a cleavable bond or an optionally substituted cleavable group;
B 1 represents a polymerase reaction blocking group;
R 1 is-OH or-H;
P 1 is H or a phosphate group;
a fifth nucleotide analog having a structural formula shown in formula III,
wherein,
Base 3 representation of
Adenine, guanine, cytosine, thymine or uracil;
B 3 represents a polymerase reaction blocking group;
R 3 is-OH or-H;
P 3 is H or a phosphate group;
wherein the first, second, third and fourth nucleotide analogs have different bases than the fifth nucleotide analog;
the first nucleotide analogue has the same base as the third nucleotide analogue;
The first nucleotide analog and the second nucleotide analog have different bases;
the first nucleotide analogue and the second nucleotide analogue have different bases from the fourth nucleotide analogue; and
the third nucleotide analogue has a different base than the fourth nucleotide analogue.
7. The mixture of nucleotide analogs according to claim 6, wherein the structure of formula (I) is one of the following:
8. the mixture of nucleotide analogs according to claim 6, wherein said first nucleotide analog has a structure represented by formula (5):
the second nucleotide analogue has a structure represented by formula (6):
the third nucleotide analogue has a structure represented by formula (2):
the fourth nucleotide analogue has a structure represented by formula (1):
the fifth nucleotide analog has a structure represented by formula (7):
9. a kit, comprising:
the nucleotide analog mixture of claim 6.
10. The kit of claim 9, further comprising:
a cleaving reagent that can act on a cleavable group or a cleavable bond.
11. A method of performing a polymerase reaction comprising:
(1) Placing a mixture comprising a single-stranded template, a primer, the nucleotide analog mixture of claim 6, and a polymerase under conditions suitable for primer extension,
wherein the primer matches a portion of the single stranded template such that only one first new nucleotide extends at the 3' end of the primer.
12. The method of claim 11, wherein the single stranded template or primer is immobilized on a solid support.
13. The method of claim 12, wherein the single stranded template or primer is immobilized on a chip.
14. The method as recited in claim 11, further comprising:
(2) Cleaving the polymerase reaction blocking group of the first new nucleotide and returning to step (1) to continue extending only one second new nucleotide at the 3' end of the ribose or deoxyribose sugar of the first new nucleotide.
15. The method of claim 11, further comprising, after step (1):
(1-1) detecting a fluorescent signal and a phosphorescent signal of the first new base, respectively.
16. The method of claim 15, further comprising, prior to step (1-1): removing the reactants from the system after extending the first new nucleotide.
17. The method of claim 15, further comprising, after step (1-1):
(1-2) determining the type of at least one of the first new base and the base at the position on the single-stranded template corresponding to the first new base based on at least one of the fluorescent signal and the phosphorescent signal.
18. The method of claim 17, wherein the fluorescent signal and the phosphorescent signal are present together at a base indicating the first new nucleotide as the base corresponding to the first nucleotide analogue and the third nucleotide analogue,
the absence of both the fluorescent signal and the phosphorescent signal indicates that the base of the first new nucleotide is the base corresponding to the fifth nucleotide analog,
the presence of the fluorescent signal, but the absence of the phosphorescent signal, indicates that the base of the first new nucleotide is the base corresponding to the second nucleotide analog,
the fluorescent signal is absent, but the presence of the phosphorescent signal indicates that the first new nucleotide is the base to which the fourth nucleotide analog corresponds.
19. The method of claim 17, wherein in step (1-2), an excitation wavelength of 400 to 480nm and a signal acquisition filter of 500 to 570nm are used.
20. The method of claim 17, wherein the fluorescent signal is collected during a time period when the excitation light is on and the phosphorescent signal is collected between 0.5 and 100 milliseconds after the excitation light is off.
21. A method for determining a nucleic acid sequence, comprising: performing a controlled chain polymerase reaction on a test nucleic acid sequence using the method of any one of claims 11 to 20.
22. A sequencer, comprising:
a housing;
a chain polymerase reaction region disposed in the housing;
an excitation light emitter adapted to emit excitation light of a predetermined wavelength toward the chain polymerase reaction region;
a signal acquisition device adapted to acquire fluorescent and phosphorescent signals in the chain polymerase reaction region; and
the controller is connected with the signal acquisition device and the laser emitter respectively and is used for controlling the on and off of the excitation light emitter and controlling the signal acquisition device to switch between acquiring fluorescent signals and phosphorescent signals, and the controller is suitable for controlling the signal acquisition device to acquire the fluorescent signals in the process of starting the excitation light emitter and acquiring the phosphorescent signals in a preset time range after the excitation light emitter is closed.
23. The sequencer according to claim 22, wherein said signal acquisition means comprises:
a camera; and
the filter plate has a filter range of 500-570 nm.
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