CN112601825A - Nucleic acid sequencing method - Google Patents
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Abstract
The invention provides a nucleic acid sequencing method and a corresponding sequencing device, and the method is characterized in that a nucleotide analogue with fluorescent and phosphorescent groups is merged into the 3 'end of an initial growing 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
PRIORITY INFORMATION
None.
The invention relates to the field of biomedicine, in particular to a nucleic acid sequencing method, nucleotide analogs, a nucleotide analog mixture and application thereof.
The DNA sequencing technology comprises a first generation DNA sequencing technology represented by a Sanger (Sanger) sequencing method and a second generation DNA sequencing technology represented by Illumina Hiseq2500, Roche 454, ABI Solid, BGISEQ-500 and the like. The Sanger sequencing method has the characteristics of simple experimental operation, intuitive and accurate result, short experimental period and the like, and is widely applied to the fields of clinical gene mutation detection, genotyping and the like with high requirements on timeliness of detection results. However, sanger sequencing has the disadvantages of low throughput and high cost, which limits 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 sequencing technology of Hiseq2500V2 as an example, one experimental flow can generate data of 10-200G bases, the average sequencing cost of each base is less than 1/1000 of the sequencing cost of Sanger sequencing method, and the obtained sequencing result can be directly processed and analyzed by a computer. Therefore, second generation DNA sequencing technologies are well suited for large scale sequencing.
Currently, the second generation DNA sequencing technology mainly involves Sequencing By Ligation (SBL) technology and Sequencing By Synthesis (SBS) technology. Typical examples of such sequencing techniques include SOLID sequencing developed by Applied Biosystems, Combined Probe Anchor ligation developed by Complete Genomics (cPAL) and Combined Probe Anchor Synthesis developed by Wasabgene (cPAS), Illumina sequencing developed by Illumina and Solexa technology, and the like. Among these sequencing methods, Illumina and compate Genomics use a method of detecting optical signals, and in order to achieve the identification and discrimination of 4 bases (A, T/U, C and G), it is usually necessary to label the 4 bases with 4 fluorescent dyes, respectively. In this case, in order to read the fluorescence signal carried by each base, the sequencing device must be equipped with at least 2 monochromatic excitation light sources and at least 2 cameras, which results in the sequencing device being expensive and bulky to manufacture.
It has been reported that identification and discrimination of 4 bases can be achieved by using 2 fluorescent dyes (Sara Goodwin, et al. Nature Reviews Genetics 17,333-351 (2016)). For example, NextSeq sequencing systems and Mini-Seq sequencing systems developed by Illumina Inc. use dual fluorescent dye-based sequencing methods. In such sequencing methods, the identification and discrimination 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 not base T/U. In such sequencing methods, the sequencing apparatus requires only one camera, but still needs to be equipped with at least 2 monochromatic excitation light sources. Therefore, the manufacturing cost and volume of the sequencing device using 2 fluorescent dyes are still relatively high. In addition, the sequencing quality of the dual fluorescent dye-based sequencing method is significantly reduced compared to the sequencing method using 4 fluorescent dyes, mainly because the difficulty in distinguishing the dual-color fluorescence from the single-color fluorescence is large and the accuracy is reduced. While a sequencing method using a fluorescent material, such as illumina iseq100, requires a special reagent to be added to a sequencing chip to realize signal transformation during the first and second signal acquisition periods so as to distinguish different bases, so that the sequencing process is more complicated and longer in duration.
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 interaction, and when the signal conversion is realized by using different chemical excision reactions and biotin/streptavidin interaction, reaction reagents are added between the first image acquisition and the second image acquisition, so that the biochemical complexity of sequencing is increased, the sequencing time is prolonged, and the reagent cost is increased due to the fact that certain signals are added by using small molecules and protein interaction. However, in the two-color fluorescence sequencing method in the prior art, two kinds of fluorescence are used for distinguishing four bases, and the method needs 2 kinds of laser to excite two fluorescent molecules with different excitation wavelengths, so that the miniaturization development of an instrument is not facilitated, and the cost of the instrument cannot be well reduced.
Based on the above facts and findings, the inventors propose for the first time a principle of using the difference of optical properties of compounds to distinguish bases, and a sequencer based on the principle only needs one kind of excitation light and one lens of a filter device, and does not need to perform a chemical reaction between two photographs, thereby simplifying the hardware conditions and the sequencing steps of the sequencer in principle, and being beneficial to reducing the cost of the instrument and the cost of reagents. Meanwhile, the inventor also designs a new nucleotide analogue and a nucleotide analogue mixture, and can conveniently, quickly and accurately distinguish basic groups by utilizing the difference of the optical properties of the nucleotide analogue mixture so as to realize DNA and/or RNA sequencing.
In a first aspect of the invention, the invention features a method of sequencing a nucleic acid molecule. According to an embodiment of the invention, the method comprises: (1) subjecting a nucleic acid molecule to be tested to an annealing reaction with a primer so as to form an initial duplex, said nucleic acid molecule or primer to be tested being immobilized beforehand on a support, said duplex consisting of said nucleic acid molecule to be tested and said primer, said duplex being immobilized on said support; (2) incorporating one or both of first through fifth nucleotide analogs into the 3 'end of said growing nucleic acid strand under the catalytic action of a polymerase with the primer in said duplex being a first growing nucleic acid strand, such that only a first new nucleotide extends at the 3' end of said growing nucleic acid strand to form a first product duplex; (3) removing the polymerase and the unreacted first to fifth nucleotide analogues in the reaction systems in the steps (1) and (2); (4) judging the first nucleotide at the 3' end of the nucleic acid molecule to be detected based on the fluorescence signal and the phosphorescence signal of the first product duplex; wherein the first to fifth nucleotide analogs have base complementary pairing ability, a hydroxyl group at the 3' -position of the ribose or deoxyribose sugar of the first to fifth nucleotide analogs is protected by a protecting group which 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, the first and second nucleotide analogs have different bases from the fourth nucleotide analog, and the third and fourth nucleotide analogs have different bases, the first and second nucleotide analogs each independently carry a fluorescent group and no phosphorescent group, the third and fourth nucleotide analogs each independently carry a phosphorescent group and no fluorescent group, and the fifth nucleotide analog does not carry a fluorescent group and a phosphorescent group. Based on the principle that the difference of optical properties of compounds is used for distinguishing bases, the inventors designed a novel nucleic acid sequencing method, and the corresponding bases in the nucleic acid molecules to be sequenced are identified by using the optical signals (with phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence) of the first to fifth nucleotide analogs (A, T/U, C and G). With the sequencing method according to an embodiment of the present invention, only one excitation light and one filtering device are required, and no compound reaction is required between two detection signals. Therefore, the sequencing steps are simplified, the sequencing cost is reduced, and the sequencing result is accurate.
In a second aspect of the invention, a nucleotide analog is provided. According to an embodiment of the invention, the nucleotide analogue has a structural formula shown in formula (I),
wherein, Base1Represents adenine, guanine, cytosine, thymine or uracil; d1Represents a phosphorescent group; c1Represents a cleavable bond or optionalA substituted cleavable group; b is1Represents a polymerase reaction blocking group; r1is-OH or-H, P1Is H or a phosphate group. The inventor firstly proposes and designs the nucleotide analogue carrying the phosphorescent group, the nucleotide analogue carrying the phosphorescent group can be independently used in DNA and/or RNA sequencing, the nucleotide analogue carrying the fluorescent group in the sequencing method of the fluorescent dye in the prior art such as a bicolor fluorescence sequencing method, a monochromatic fluorescence sequencing method and the like is replaced, and meanwhile, the sequencing method for distinguishing the base by utilizing the optical property difference of the nucleotide analogue carrying the phosphorescent group and the nucleotide analogue carrying the fluorescent group, which is firstly proposed by the application, can be realized. The nucleotide analogue according to the embodiment of the invention 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 mixture of nucleotide analogs comprises the nucleotide analogs described above. The inventors propose for the first time a nucleotide analogue mixture comprising nucleotide analogues carrying phosphorescent groups, which in turn can be used to generate phosphorescent optical signals to effect sequencing of nucleic acids.
In a fourth aspect of the invention, the invention features a mixture of nucleotide analogs. According to an embodiment of the invention, the mixture of nucleotide analogues comprises: a first nucleotide analog and a second nucleotide analog, each independently having a structural formula shown in formula II,
a third nucleotide analog and a fourth nucleotide analog, each independently having a structural formula shown in formula I,
a fifth nucleotide analog having a structural formula shown in formula III,
wherein the first, second, third, and fourth nucleotide analogs have a different base than the fifth nucleotide analog; the first nucleotide analog and the third nucleotide analog have the same base; the first nucleotide analog and the second nucleotide analog have different bases; the first nucleotide analog and the second nucleotide analog have different bases from the fourth nucleotide analog; and the third nucleotide analog and the fourth nucleotide analog have different bases. The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, based on the principle, the inventor designs a nucleotide analogue mixture, uses the first to fifth nucleotide analogues to carry out nucleic acid sequencing, quickly and accurately identifies the type of the corresponding base on a nucleic acid chain by detecting different optical signals (simultaneously having phosphorescence and fluorescence, only phosphorescence and no fluorescence, only fluorescence and no phosphorescence, and no phosphorescence and fluorescence).
In a fifth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises: a nucleotide analogue as hereinbefore described, or a mixture of nucleotide analogues as hereinbefore described. The inventor firstly proposes and designs the nucleotide analogue carrying the phosphorescent group, 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 used in the fluorescent dye sequencing method in the prior art, such as a bicolor fluorescent sequencing method, a monochromatic fluorescent sequencing method and the like. Meanwhile, the sequencing method for distinguishing bases by utilizing the optical property difference of the compound, which is firstly proposed by the application, can be realized by utilizing the optical property difference of the nucleotide analogue carrying the phosphorescent group and the fluorescent group analogue, so that the nucleotide analogue mixture is designed and applied to the preparation of the kit. The kit provided by the embodiment of the invention has the advantages of low cost and simplicity and convenience in operation, and can be used for quickly and accurately detecting the type of the basic group in the nucleic acid molecule.
In a sixth aspect of the invention, a method of performing a polymerase reaction is presented. According to an embodiment of the invention, the method comprises: (1) placing a mixture comprising a single-stranded template, a primer, a mixture of nucleotide analogs as described above, and a polymerase under conditions suitable for primer extension, wherein the primer is matched to a portion of the single-stranded template such that only a first new nucleotide is extended at the 3' end of the primer. The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, and based on the principle, the inventor designs a nucleotide analogue mixture, uses the first to fifth nucleotide analogues to perform nucleic acid sequencing, and accurately identifies the type of the corresponding base in a nucleic acid chain by detecting different optical signals (simultaneously with phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence). According to the method provided by the embodiment of the invention, the nucleotide analogue can be accurately paired with the nucleic acid chain, so that the nucleic acid sequencing can be conveniently, quickly and accurately carried out.
In a seventh aspect of the invention, a method of nucleic acid sequencing is provided. 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 above. 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 a chemical reaction is not needed between two times of photographing, so that the sequencing step is simplified, the sequencing time is shortened, the sequencing cost is reduced, and the sequencing result is accurate.
In an eighth aspect of the invention, a sequencer is provided. According to an embodiment of the invention, the sequencer comprises: 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 a fluorescent signal and a phosphorescent signal in the chain polymerase reaction region. The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, based on the principle, the inventor designs a nucleotide analogue mixture, utilizes the first to fifth nucleotide analogues to carry out nucleic acid sequencing, and identifies the types of the bases corresponding to nucleic acid chains by detecting different optical signals (simultaneously having phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence). 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.
FIG. 1 is a sequencer according to an embodiment of the invention;
FIG. 2 is a sequencer (including a controller) according to an embodiment of the 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.
Reference numerals:
the sequencing device comprises a sequencer 1000, a shell 100, a chain polymerase reaction area 11, a laser emitter 12, a signal acquisition device 13 and a controller 14.
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 with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Method for sequencing nucleic acid molecules
In a first aspect of the invention, the invention features a method of sequencing a nucleic acid molecule. According to an embodiment of the invention, the method comprises: (1) subjecting a nucleic acid molecule to be tested to an annealing reaction with a primer so as to form an initial duplex, said nucleic acid molecule or primer to be tested being immobilized beforehand on a support, said duplex consisting of said nucleic acid molecule to be tested and said primer, said duplex being immobilized on said support; (2) incorporating one or both of first through fifth nucleotide analogs into the 3 'end of said growing nucleic acid strand under the catalytic action of a polymerase with the primer in said duplex being a first growing nucleic acid strand, such that only a first new nucleotide extends at the 3' end of said growing nucleic acid strand to form a first product duplex; (3) removing the polymerase and the unreacted first to fifth nucleotide analogues in the reaction systems in the steps (1) and (2); (4) judging the first nucleotide at the 3' end of the nucleic acid molecule to be detected based on the fluorescence signal and the phosphorescence signal of the first product duplex; wherein the first to fifth nucleotide analogs have base complementary pairing ability, a hydroxyl group at the 3' -position of the ribose or deoxyribose of the first to fifth nucleotide analogs is protected by a protecting group which 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, the first and second nucleotide analogs have different bases from the fourth nucleotide analog, the third and fourth nucleotide analogs have different bases, the first and second nucleotide analogs each independently carry a fluorescent group and no phosphorescent group, the third and fourth nucleotide analogs each independently carry a phosphorescent group and no fluorescent group, and the fifth nucleotide analog does not carry a fluorescent group and a phosphorescent group. Based on the principle that the difference of optical properties of compounds is used for distinguishing bases, the inventors designed a novel nucleic acid sequencing method, and the corresponding bases in the nucleic acid molecules to be sequenced are identified by using the optical signals (with phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence) of the first to fifth nucleotide analogs (A, T/U, C and G). With the sequencing method according to an embodiment of the present invention, only one excitation light and one filtering device are required, and no compound reaction is required between two detection signals. 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 said first product duplex to a cleavage treatment in a reaction system comprising a solution phase and a solid phase to remove the protecting group and/or the light signaling group at the 3' position of the ribose or deoxyribose sugar in said 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 said growing nucleic acid strand under the catalytic action of a polymerase with the cleavage treatment product in the reaction system of step (6) being a second growing nucleic acid strand so as to extend only one second new nucleotide at the 3' end of said second growing nucleic acid strand to form a second product duplex; (8) and (4) repeating the steps (3) and (4) to judge the second nucleotide sequence of the 3' end of the nucleic acid molecule to be detected. The protection group at the 3' position on the nucleotide analogue is cut to ensure the next round of the polymer chain reaction, and the luminescent group (fluorescent group or phosphorescent group) on the nucleotide analogue is cut to better detect the optical signal carried by the nucleotide analogue amplified in the next round of the polymerase chain reaction. The solution phase in the present invention refers to a reaction solution in which a reaction occurs, and the solid phase refers to a support on 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 fluorophore.
According to an embodiment of the invention, the extended first new nucleotide is a third and a fourth nucleotide analogue and the cleaved light signaling group is a phosphorescent group.
According to an embodiment of the invention, the extended first new nucleotide is a fifth nucleotide analogue that cleaves only the protecting group at the 3' position of the ribose or deoxyribose sugar in said protecting group.
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 the steps (5) to (8) one or more times to determine the nucleotide sequence of the nucleic acid molecule to be detected. Repeating the steps (5) - (8) one or more times until the sequence of the complete segment of the nucleic acid molecule to be sequenced is determined.
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 analog and the third nucleotide analog, 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 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 analog. Therefore, bases corresponding to the nucleic acid molecules to be sequenced can be rapidly and accurately determined through different optical signals.
According to an embodiment of the present invention, the nucleic acid molecule to be detected is DNA, and the nucleic acid molecule to be detected is subjected to denaturation treatment in advance to obtain a single-stranded nucleic acid molecule to be detected.
Nucleotide analogs
In a second aspect of the invention, a nucleotide analog is provided. According to an embodiment of the invention, the nucleotide analogue has a structural formula shown in formula (I),
wherein, Base1Represents adenine, guanine, cytosine, thymine or uracil; d1Represents a phosphorescent group; c1Represents a cleavable bond or an optionally substituted cleavable group; b is1Represents a polymerase reaction blocking group; r1is-OH or-H, P1Is H or a phosphate group. The inventor firstly proposes and designs the nucleotide analogue carrying the phosphorescent group, and the nucleotide analogue carrying the phosphorescent groupThe method can be independently used for DNA and/or RNA sequencing, replaces the nucleotide analogs carrying fluorescent groups in the fluorescent dye sequencing methods used in the prior art such as a two-color fluorescence sequencing method, a single-color fluorescence sequencing method and the like, and can realize the sequencing method for distinguishing the base groups by using the optical property difference of the nucleotide analogs by using the difference of the optical properties of the nucleotide analogs carrying phosphorescent groups and the optical property difference of the nucleotide analogs carrying fluorescent groups. The nucleotide analogue according to the embodiment of the invention 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, and may be a group that emits a phosphorescent signal that can be detected controllably under a specific excitation light. For example, the phosphorescent group may be of the following structure:
wherein, R is4Is H, F, Cl, Br, I, CN, NO2、C 1-6Alkyl radical, C1-6Haloalkyl, C1-6An alkoxy group. The above groups can phosphoresce under the action of specific excitation light.
According to an embodiment of the invention, the phosphate group is a monophosphate group, a diphosphate group, a triphosphate group or a polyphosphate group.
It is to be noted that the cleavable group is not particularly limited as long as it can be cleaved under specific conditions. For example, the cleavable group may be a group comprising-S-S-, CH2CH＝CH 2、-CH 2N 3And cleavable groups common in the prior art.
According to an embodiment of the invention, said C1Further comprising a polymerase reaction blocking group. It is to be noted that if the above-mentioned cleavable group includes a site for polymerase reaction, it is necessaryIntroducing a blocking group for polymerase reaction at the site of polymerase reaction to prevent reaction at the site during polymerase chain reaction or sequencing from affecting sequencing results.
For example, the C1Including but not limited to the following structures:
wherein n 1-n 17 are each independently an integer of 0-7.
It should be noted that 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 including-S-S-, CH2CH＝CH 2、-CH 2N 3And the like, polymerase reaction blocking groups commonly found in the prior art. The group can control only one new nucleotide to be amplified each time in the polymerase chain reaction or sequencing process, and then after an optical signal is detected, the group is cut off and then the next round of amplification is carried out.
According to an embodiment of the present invention, the structure represented by formula (I) is a structure of 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 mixture of nucleotide analogs comprises the nucleotide analogs described above. The inventors propose for the first time a nucleotide analogue mixture comprising nucleotide analogues carrying phosphorescent groups, which in turn can be used to generate phosphorescent optical signals to effect sequencing of nucleic acids.
Nucleotide analogue mixture
In a fourth aspect of the invention, the invention features a mixture of nucleotide analogs. According to an embodiment of the invention, the mixture of nucleotide analogues comprises: a first nucleotide analog and a second nucleotide analog, each independently having a structural formula shown in formula II,
a third nucleotide analog and a fourth nucleotide analog, each independently having a structural formula shown in formula I,
a fifth nucleotide analog having a structural formula shown in formula III,
wherein the first, second, third, and fourth nucleotide analogs have a different base than the fifth nucleotide analog; the first nucleotide analog and the third nucleotide analog have the same base; the first nucleotide analog and the second nucleotide analog have different bases; the first nucleotide analog and the second nucleotide analog have different bases from the fourth nucleotide analog; and the third nucleotide analog and the fourth nucleotide analog have different bases. The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, based on the principle, the inventor designs a nucleotide analogue mixture, uses the first to fifth nucleotide analogues to carry out nucleic acid sequencing, quickly and accurately identifies the type of the corresponding base on a nucleic acid chain by detecting different optical signals (simultaneously having phosphorescence and fluorescence, only phosphorescence and no fluorescence, only fluorescence and no phosphorescence, and no phosphorescence and fluorescence).
The phosphorescent group is not particularly limited, and may be a group that emits a phosphorescent signal that can be detected controllably under a specific excitation light. For example, the phosphorescent group may be of the following structure:
wherein, R is4Is H, F, Cl, Br, I, CN, NO2、C 1-6Alkyl radical, C1-6Haloalkyl, C1-6An alkoxy group. The above groups can phosphoresce under the action of specific excitation light.
According to an embodiment of the invention, the phosphate group is a monophosphate group, a diphosphate group, a triphosphate group or a polyphosphate group.
It is to be noted that the cleavable group is not particularly limited as long as it can be cleaved under specific conditions. For example, the cleavable group may be a group comprising-S-S-, CH2CH＝CH 2、-CH 2N 3And cleavable groups common in the prior art.
According to an embodiment of the invention, said C1And/or C2Further comprising a polymerase reaction blocking group. It should be noted that, if the cleavable group includes a site for polymerase reaction, a blocking group for polymerase reaction needs to be introduced at the site for polymerase reaction, so as to prevent the polymerase chain reaction or the reaction at the site during the sequencing process from affecting the sequencing result.
For example, the C1And/or C2Including but not limited to the following structures:
wherein n 1-n 17 are each independently an integer of 0-7.
It is to be noted that the polymerase reaction blocking group is not particularly limited as long as it can block the polymerase reaction under specific conditionsThe site is accessible for polymerase reaction and, under another specific condition, can be removed to allow polymerase reaction to proceed. For example, the polymerase reaction blocking group may be a group including-S-S-, CH2CH＝CH 2、-CH 2N 3And the like, polymerase reaction blocking groups commonly found in the prior art. The group can control only one new nucleotide to be amplified each time in the polymerase chain reaction or sequencing process, and then after an optical signal is detected, the group is cut off and then the next round of amplification is carried out.
The fluorescent group is not particularly limited, and may be any fluorescent group that can emit a fluorescence signal that can be controllably detected under the condition of a specific excitation light. E.g. D2Including but not limited to the following structures:
according to an embodiment of the present invention, the structure represented by formula (I) is a structure of one of the following:
according to a specific embodiment of the present invention, the first nucleotide analog has a structure represented by formula (5):
the second nucleotide analog has a structure represented by formula (6):
the third nucleotide analog has a structure represented by formula (2):
the fourth nucleotide analog has a structure represented by formula (1):
the fifth nucleotide analog has a structure represented by formula (7):
Reagent kit
In a fifth aspect of the invention, a kit is provided. According to an embodiment of the invention, the kit comprises: a nucleotide analogue as hereinbefore described, or a mixture of nucleotide analogues as hereinbefore described. The inventor firstly proposes and designs the nucleotide analogue carrying the phosphorescent group, 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 used in the fluorescent dye sequencing method in the prior art, such as a bicolor fluorescent sequencing method, a monochromatic fluorescent sequencing method and the like. Meanwhile, the sequencing method for distinguishing bases by utilizing the optical property difference of the compound, which is firstly proposed by the application, can be realized by utilizing the optical property difference of the nucleotide analogue carrying the phosphorescent group and the fluorescent group analogue, so that the nucleotide analogue mixture is designed and applied to the preparation of the kit. The kit provided by the embodiment of the invention has the advantages of low cost and simplicity and convenience in operation, and can be used for quickly and accurately detecting the type of the basic group in the nucleic acid molecule.
According to an embodiment of the invention, the kit further comprises: a cleavage reagent that can act on the cleavable group or cleavable bond. The cleavable group or cleavable bond in the nucleotide analog or nucleotide analog mixture can be cleaved by the cleavage reagent, so that the polymerase reaction blocking group is removed to expose the site of polymerase reaction for the next new base amplification.
It should be noted that the cleavage reagent is not particularly limited as long as it can cleave the cleavable bond or 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 a disulfide bond efficiently, and an organic phosphine compound can be used as a cleavage reagent to cleave an azide group efficiently.
Method for polymerase reaction
In a sixth aspect of the invention, a method of performing a polymerase reaction is presented. According to an embodiment of the invention, the method comprises: (1) placing a mixture comprising a single-stranded template, a primer, a mixture of nucleotide analogs as described above, and a polymerase under conditions suitable for primer extension, wherein the primer is matched to a portion of the single-stranded template such that only a first new nucleotide is extended at the 3' end of the primer. The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, and based on the principle, the inventor designs a nucleotide analogue mixture, uses the first to fifth nucleotide analogues to perform nucleic acid sequencing, and accurately identifies the type of the corresponding base in a nucleic acid chain by detecting different optical signals (simultaneously with phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence). According to the method provided by the embodiment of the invention, the nucleotide analogue can be accurately paired with the nucleic acid chain, so that the nucleic acid sequencing can be conveniently, quickly and accurately carried out.
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 extending only one second new base at the 3' end of the first new nucleotide. The method is beneficial to accurately and quickly sequencing the nucleic acid molecules.
According to an embodiment of the present invention, after the step (1), further comprising: (1-1) detecting the fluorescent signal and the 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 the step (1-1), further comprising: removing the reactant from the system after extending the first new nucleotide. By removing the reactant in the system after the first new nucleotide is extended and then carrying out optical signal detection, the sensitivity and the accuracy of optical signal detection can be improved.
According to an embodiment of the present invention, after the 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 corresponding to the first new base on the single-stranded template based on at least one of the fluorescent signal and the phosphorescent signal.
According to an embodiment of the present invention, the presence of the fluorescent signal and the phosphorescent signal simultaneously indicates that the base of the first new nucleotide is a base corresponding to the first nucleotide analogue and the third nucleotide analogue, the absence of the fluorescent signal and the phosphorescent signal indicates that the base of the first new nucleotide is a base corresponding to the fifth nucleotide analogue, the presence of the fluorescent signal but the absence of the phosphorescent signal indicates that the base of the first new nucleotide is a base corresponding to the second nucleotide analogue, the absence of the fluorescent signal but the presence of the phosphorescent signal indicates that the base of the first new nucleotide is a base corresponding to the fourth nucleotide analogue. Therefore, 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 the embodiment of the invention, in the step (1-2), an excitation wavelength of 400-480 nm and a signal collection filter of 500-570 nm are adopted. The inventor finds that on the basis of the excitation wavelength and the signal acquisition filter, the fluorescence signal and the phosphorescence signal can be detected successively, the detection requirement can be met only by one excitation light and one signal acquisition filter, and meanwhile, no chemical reaction is needed in the two signal acquisition processes. Thus, sequencing time and sequencing cost are saved.
According to the embodiment of the invention, the fluorescence signal is collected in the time period when the exciting light is on, and the phosphorescence signal is collected between 0.5-100 milliseconds after the exciting light is off. The method of the embodiment of the invention is used for carrying out polymerase reaction, no chemical reaction is needed to be carried out between two signal acquisition processes, and the time interval of the two signal acquisition processes is short. Thus, sequencing time and sequencing cost are saved.
Method for determining nucleic acid sequence
In a seventh aspect of the invention, a method of nucleic acid sequencing is provided. 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 above. 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 a chemical reaction is not needed between two times of photographing, so that the sequencing step is simplified, the sequencing time is shortened, the sequencing cost is reduced, and the sequencing result is accurate.
Sequencing instrument
In a sixth aspect of the invention, a sequencer is provided. According to an embodiment of the present invention, referring to fig. 1, 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, the laser emitter 12 being adapted to emit excitation light of a predetermined wavelength to the chain polymerase reaction region 11; according to an embodiment of the invention, the predetermined wavelength is 400-480 nm.
And a signal acquisition device 13, the signal acquisition device 13 being adapted to acquire a fluorescent signal and a phosphorescent signal in the chain polymerase reaction area 11.
According to 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, and is used for controlling the excitation light emitter 12 to be turned on and off and controlling the signal acquisition device 13 to switch between acquiring a fluorescence signal and acquiring a phosphorescence signal. According to a further embodiment of the present invention, the controller 14 is adapted to control the signal collecting device 13 to collect a fluorescence signal during the start-up of the excitation light emitter 12 and to collect a phosphorescence signal within a predetermined time range after the shut-down of the excitation light emitter 12. According to still another embodiment of the present invention, the predetermined time is 0.5 to 100 milliseconds.
The inventor firstly proposes to use the difference of optical properties of compounds to distinguish bases, based on the principle, the inventor designs a nucleotide analogue mixture, utilizes the first to fifth nucleotide analogues to carry out nucleic acid sequencing, and identifies the types of the bases corresponding to nucleic acid chains by detecting different optical signals (simultaneously having phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and no fluorescence). 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.
Preparation method
The compounds represented by the formulae (II) and (III) in the nucleotide analogue mixture referred to in the present application are nucleotide analogues commonly known in the art, and the synthesis can be performed by referring to the methods in the art, for example, the methods in WO 2017/058953A1, WO 2017/087887A1, US 2017/0130051A1 and the like.
The compound shown in the formula (I) in the nucleotide analogue mixture is the nucleotide analogue which is proposed by the inventor for the first time, and the synthesis method can also refer to the method in the prior art, except that the compound needs to be synthesized by converting a fluorescent group into a phosphorescent group in the synthesis process. The synthesis of phosphorescent groups can be carried out by reference to the methods for the synthesis of relevant phosphorescent groups known in the art, for example, the methods in Fraser, C.L., NATURE MATERIALS,2009,08,747-751(DOI:10.1038/NMAT 2509).
For example, the following starting material 1 can be obtained by the method described in Fraser, C.L., NATURE MATERIALS,2009,08,747-751(DOI:10.1038/NMAT2509) literature:
obtaining a raw material 2 with a fluorescent group to be connected (in the invention, the raw material 2 can be considered as a raw material 2 with a phosphorescent group to be connected) by using the methods in WO 2017/058953A1, WO 2017/087887A1, US 2017/0130051A1 and the like;
then, the raw material 1 and the raw material 2 to be connected with the phosphorescent group are synthesized by classical chemical reactions such as condensation reaction, substitution reaction or addition reaction under appropriate conditions, so as to obtain the nucleotide analogue carrying the phosphorescent group.
Example 1:
the inventors verified the sequencing method by using the following compounds:
the optical signals carried by the above compounds are shown in table 1 below:
table 1:
| base type | Fluorescent channel | Phosphorescent channels |
| A | Luminescence | Does not emit light |
| G | Does not emit light | Does not emit light |
| C | Does not emit light | Luminescence |
| T | Luminescence | Luminescence |
In the method, a mixture of phosphorescence-modified and fluorescence-modified dTTP is used as the T base.
In order to verify the base discrimination based on this principle, several DNAs having different sequences were immobilized on a glass chip by a spotting instrument under a fluorescence microscope. The diameter of each point is 50 microns, the excitation wavelength of the fluorescence microscope is selected to be in the range of 400-480 nanometers, and the range of the fluorescence and phosphorescence acquisition signal filter is in the range of 500-570 nanometers. The fluorescent signal is collected during the time period when the excitation light is on, and the phosphorescent signal is collected between 0.5ms and 100ms after the excitation light is off.
In the verification experiment, the enzyme and various buffers in the BGISEQ-500 reagent are adopted as the reagents. Wherein the fluorescent modified probe of the sequencing reagent is changed into the 5 fluorescent and phosphorescent modified reversible blocking nucleotides.
First, a DNA array (boao biochip customization service) on a silicon wafer was purchased, four DNAs having the same sequence were used, and four bases were contained in the four sequences at each position corresponding to the region to be detected, wherein the four sequences are shown in table 2 below.
Table 2:
after the DNA is fixed, a sequencing primer of partially complementary sequence is added (as in Table 2 above). After adding a DNA polymerase mixture containing the above five nucleotides to a silicon chip, the 3' end of each DNA primer is polymerized with the corresponding base under PCR extension conditions, then unreacted nucleotides are washed away, 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 signal acquisition time of the microscope.
The first cycle first obtained figure 3, images of the fluorescent channel on the left and the phosphorescent channel on the right, where the two leftmost columns were the mixed DNA localization areas, so 6 spots all had signals on both channels, 3-10 columns were single species DNA sequence spots, for a total of 6 × 8 spots.
After 5 cycles of sequencing, the sequences of the 48 points are respectively judged to be;
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 completely the same as the sequence designed by the custom chip, and the reading length of the short sequencing reaches 100 percent of accuracy.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean 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 invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer 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, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (26)
- A method of sequencing a nucleic acid molecule,(1) subjecting a nucleic acid molecule to be tested to an annealing reaction with a primer so as to form an initial duplex, said nucleic acid molecule or primer to be tested being immobilized beforehand on a support, said duplex consisting of said nucleic acid molecule to be tested and said primer, said duplex being immobilized on said support;(2) incorporating one or both of first through fifth nucleotide analogs into the 3 'end of said growing nucleic acid strand under the catalytic action of a polymerase with the primer in said duplex being a first growing nucleic acid strand, such that only a first new nucleotide extends at the 3' end of said growing nucleic acid strand to form a first product duplex;(3) removing the polymerase and the unreacted first to fifth nucleotide analogues in the reaction systems in the steps (1) and (2);(4) judging the first nucleotide at the 3' end of the nucleic acid molecule to be detected based on the fluorescence signal and the phosphorescence 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 the ribose or deoxyribose of the first to fifth nucleotide analogs is protected by a protecting group which is a polymerase reaction blocking group,the first, second, third, and fourth nucleotide analogs have a different base than the fifth nucleotide analog,the first nucleotide analog and the third nucleotide analog have the same base,the first nucleotide analog and the second nucleotide analog have different bases,the first nucleotide analog and the second nucleotide analog have different bases from the fourth nucleotide analog,the third nucleotide analog has a different base from the fourth nucleotide analog,the first and second nucleotide analogs each independently carry a fluorescent group and do not carry a phosphorescent group,the third and fourth nucleotide analogs each independently carry a phosphorescent group and no fluorescent group,the fifth nucleotide analogue does not carry a fluorescent group and a phosphorescent group.
- The method of claim 1, further comprising:(5) subjecting the first product duplex to a cleavage treatment in a reaction system comprising a solution phase and a solid phase to remove the protecting group and/or the optical signaling group at the 3' -position of ribose or deoxyribose sugar in the nucleotide analogue,(6) removing the solution phase of the reaction system in the step (5),(7) incorporating one or both of the first through fifth nucleotide analogs into the 3 'end of the growing initial nucleic acid strand catalyzed by the polymerase with the cleavage treatment product in the reaction system of step (6) as the second growing nucleic acid strand such that only one second new nucleotide extends at the 3' end of the second growing initial nucleic acid strand to form a second product duplex;(8) and (4) repeating the steps (3) and (4) to judge the second nucleotide sequence of the 3' end of the nucleic acid molecule to be detected.
- The method of claim 2 wherein the extended first new nucleotide is a first or second nucleotide analog and the cleaved light signaling group is a fluorophore;optionally, the extended first new nucleotide is a third and a fourth nucleotide analog, 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 the ribose or deoxyribose sugar in the protecting group.
- The method according to claim 2, characterized in that the method further comprises the following step (9):(9) repeating the steps (5) to (8) one or more times to determine the nucleotide sequence of the nucleic acid molecule to be detected.
- The method of claim 1 to 4, wherein the simultaneous presence of the fluorescent signal and the phosphorescent signal indicates that the bases corresponding to the nucleic acid molecule to be sequenced are the bases corresponding to the first nucleotide analog and the third nucleotide analog,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;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 to obtain a single-stranded nucleic acid molecule to be detected.
- A nucleotide analogue characterized by having a structural formula shown in formula (I),wherein the content of the first and second substances,
Base 1represents adenine, guanine, cytosine, thymine or uracil;D 1represents a phosphorescent group;C 1represents a cleavable bond or an optionally substituted cleavable group;B 1represents a polymerase reaction blocking group;R 1is-OH or-H;P 1is H or a phosphate group. - A mixture of nucleotide analogs comprising the nucleotide analog of claim 5.
- A mixture of nucleotide analogs comprising:a first nucleotide analog and a second nucleotide analog, each independently having a structural formula shown in formula II,wherein the content of the first and second substances,
Base 2represents adenine, guanine, cytosine, thymine or uracil;D 2represents a fluorescent group;C 2represents a cleavable bond or an optionally substituted cleavable group;B 2represents a polymerase reaction blocking group;R 2is-OH or-H;P 2represents H or a phosphate group;a third nucleotide analog and a fourth nucleotide analog, each independently having a structural formula shown in formula I,wherein the content of the first and second substances,
Base 1represents adenine, guanine, cytosine, thymine or uracil;D 1represents a phosphorescent group;C 1represents a cleavable bond or an optionally substituted cleavable group;B 1represents a polymerase reaction blocking group;R 1is-OH or-H;P 1is H or a phosphate group;a fifth nucleotide analog having a structural formula shown in formula III,wherein the content of the first and second substances,
Base 3to representAdenine, guanine, cytosine, thymine or uracil;B 3represents a polymerase reaction blocking group;R 3is-OH or-H;P 3is H or a phosphate group;wherein the first, second, third, and fourth nucleotide analogs have a different base than the fifth nucleotide analog;the first nucleotide analog and the third nucleotide analog have the same base;the first nucleotide analog and the second nucleotide analog have different bases;the first nucleotide analog and the second nucleotide analog have different bases from the fourth nucleotide analog; andthe third nucleotide analog and the fourth nucleotide analog have different bases. - The nucleotide analogue of claim 6 or the mixture of nucleotide analogues of claim 7 or the mixture of nucleotide analogues of claim 8, wherein the structure according to formula (I) is one of the following:
- the mixture of nucleotide analogs of claim 1 or 8, wherein the first nucleotide analog has a structure represented by formula (5):
the second nucleotide analog has a structure represented by formula (6):
the third nucleotide analog has a structure represented by formula (2):
the fourth nucleotide analog has a structure represented by formula (1):
the fifth nucleotide analog has a structure represented by formula (7):
- a kit, comprising:the nucleotide analog of claim 6; orA mixture of nucleotide analogs according to claim 7; orThe mixture of nucleotide analogs of claim 8.
- The kit of claim 11, further comprising:a cleavage reagent that can act on the cleavable group or cleavable bond.
- A method of performing a polymerase reaction, comprising:(1) placing a mixture comprising a single-stranded template, a primer, a mixture of nucleotide analogs of claim 8, and a polymerase under conditions suitable for primer extension,wherein the primer is matched to a portion of the single stranded template such that only a first new nucleotide is extended at the 3' end of the primer.
- The method of claim 13, wherein the single stranded template or primer is immobilized on a solid support.
- The method of claim 14, wherein the single stranded template or primer is immobilized on a chip.
- The method of claim 13, 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.
- The method of claim 13, after step (1), further comprising:(1-1) detecting the fluorescent signal and the phosphorescent signal of the first new base, respectively.
- The method of claim 17, further comprising, before step (1-1): removing the reactant from the system after extending the first new nucleotide.
- The method of claim 17, 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 corresponding to the first new base on the single-stranded template based on at least one of the fluorescent signal and the phosphorescent signal.
- The method of claim 19, wherein the presence of both the fluorescent signal and the phosphorescent signal indicates that the base of the first new nucleotide is the base corresponding to the first nucleotide analog and the third nucleotide analog,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 analogue,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 to which the second nucleotide analogue corresponds,the absence of the fluorescent signal, but the presence of the phosphorescent signal indicates that the first new nucleotide is the base to which the fourth nucleotide analog corresponds.
- The method as claimed in claim 19, wherein in step (1-2), an excitation wavelength of 400-480 nm and a signal collection filter of 500-570 nm are used.
- The method of claim 19, 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.
- 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 13 to 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; anda signal acquisition device adapted to acquire a fluorescent signal and a phosphorescent signal in the chain polymerase reaction region.
- The sequencer according to claim 24, wherein said signal acquisition device comprises:a camera; andthe filter plate, the filtering range of filter plate is 500 ~ 570 nm.
- The sequencer according to claim 24, further comprising a controller, said controller being connected to said signal acquisition device and said laser emitter for controlling said excitation light emitter to be turned on and off and for controlling said signal acquisition device to switch between acquiring a fluorescence signal and said phosphorescence signal, said controller being adapted to control said signal acquisition device to acquire a fluorescence signal during start-up of said excitation light emitter and to acquire said phosphorescence signal within a predetermined time frame after turn-off of said excitation light emitter.
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