WO2020010495A1

WO2020010495A1 - Method for nucleic acid sequencing

Info

Publication number: WO2020010495A1
Application number: PCT/CN2018/095040
Authority: WO
Inventors: 刘二凯; 陈奥; 章文蔚; 廖莎
Original assignee: 深圳华大智造极创科技有限公司
Priority date: 2018-07-09
Filing date: 2018-07-09
Publication date: 2020-01-16
Also published as: CN112601825B; CN112601825A

Abstract

The present invention provides a method for nucleic acid sequencing and a corresponding sequencing device. The method is characterized by incorporating a nucleotide analog having fluorescent and phosphorescent groups into the 3' terminus of a growth-initiating nucleic acid chain during the amplification of a nucleic acid molecule duplex to be tested to determine the nucleotides at the 3' terminus of the nucleic acid molecule to be tested by detecting fluorescence and phosphorescence signals.

Description

Nucleic acid sequencing method

Priority information

no.

Technical field

The present invention relates to the field of biomedicine, and in particular, the present invention relates to a nucleic acid sequencing method, a nucleotide analog, a nucleotide analog mixture, and applications thereof.

Background technique

DNA sequencing technology includes the first-generation DNA sequencing technology represented by Sanger sequencing method and the second-generation DNA sequencing technology represented by Illumina Hisise 2500, Roche 454, ABI Solid, BGISEQ-500 and so on. The Sanger sequencing method has the characteristics of simple experimental operation, intuitive and accurate results, and short experimental cycle. It has a wide range of applications in clinical gene mutation detection and genotyping that require high timeliness of test results. However, the disadvantages of Sanger sequencing are its small 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 throughput, low cost, high degree of automation, and single molecule sequencing. Taking the sequencing technology of Hiseq2500V2 as an example, an experimental process can generate 10-200G base data, the average cost of sequencing each base is less than 1/1000 of the sequencing cost of Sanger sequencing method, and the obtained sequencing results Processing and analysis can be done directly from a computer. Therefore, the second-generation DNA sequencing technology is very suitable for large-scale sequencing.

The second-generation DNA sequencing technology that has been developed so far mainly involves sequencing-by-ligation (SBL) technology and sequencing-by-synthesis (SBS) technology. Typical examples of these sequencing technologies include SOLiD sequencing method developed by Applied Biosystems, combined probe anchoring ligation method (cPAL) independently developed by Complete Genomics, and combined probe anchoring synthesis method (cPAS) developed by BGI, Illumina The Illumina sequencing method developed by the company and Solexa technology company and so on. Among these sequencing methods, Illumina and Complate and Genomics used the method of detecting optical signals. In order to identify and distinguish the four bases (A, T / U, C, and G), usually four fluorescent dyes are used to separately These four bases are labeled. In this case, in order to read the fluorescence signal carried by each base, the sequencing device must be equipped with at least two monochrome excitation light sources and at least two cameras, which results in the manufacturing cost of the sequencing device being expensive and bulky.

It has been reported that the identification and differentiation of four bases can be achieved by using two kinds of 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 use sequencing methods based on dual fluorescent dyes. In such sequencing methods, the four bases are identified and distinguished through different combinations of two fluorescent dyes. For example, the base A can be labeled with a first fluorescent dye, the base G with a second fluorescent dye, the base C can be labeled with both the first and second fluorescent dyes, and the base T / U is not labeled, thereby distinguishing Four bases. In this type of sequencing method, only one camera is needed for the sequencing device, but it still needs to be equipped with at least two kinds of monochrome excitation light sources. Therefore, the manufacturing cost and volume of a sequencing device using two fluorescent dyes are still relatively high. In addition, compared with sequencing methods using four fluorescent dyes, the sequencing quality of sequencing methods based on dual fluorescent dyes has significantly decreased, mainly because it is more difficult to distinguish between two-color fluorescence and single-color fluorescence, and the accuracy has decreased. . However, a sequencing method using a fluorescent material, such as illumina ISEQ100, requires special reagents to be added to the sequencing chip during the first and second signal acquisition to achieve signal transformation in order to distinguish between different bases, thus enabling the sequencing process. More complicated and longer.

Therefore, sequencing methods still need to be developed and improved.

Summary of the invention

The single fluorescence sequencing method in the prior art mainly uses different chemical excision reactions and biotin / streptavidin interactions to achieve signal conversion, while using different chemical excision reactions and biotin / streptavidin When interacting with primes to achieve signal conversion, reaction reagents were added between the first and second picture acquisitions, which increased the complexity of sequencing biochemistry and the length of sequencing, and due to the use of small molecules and Proteins interact to add certain signals and reagent costs increase. In the existing two-color fluorescence sequencing method, two kinds of fluorescence are used to distinguish four kinds of bases. This method requires two kinds of lasers to excite two fluorescent molecules with different excitation wavelengths, which is not conducive to the miniaturization of the instrument. Can not better reduce the cost of the instrument.

Based on the findings of the above facts and problems, the inventors first proposed the principle of using the differences in the optical properties of compounds to distinguish bases. A sequencer based on this principle requires only an excitation light and a lens for a filtering device, and does not require The chemical reaction between the two pictures simplifies the hardware conditions and sequencing steps of the sequencer in principle, which is beneficial to reducing the cost of the instrument and the cost of reagents. At the same time, the inventor also designed a new nucleotide analog, and also designed a nucleotide analog mixture, using the difference in optical properties of the nucleotide analog mixture can quickly and accurately distinguish bases, This enables DNA and / or RNA sequencing.

In a first aspect of the invention, the invention provides a method for sequencing a nucleic acid molecule. According to an embodiment of the present invention, the method includes: (1) annealing a nucleic acid molecule to be tested with a primer so as to form an initial duplex, the nucleic acid molecule or primer to be tested is previously fixed on a support, so The duplex is composed of the nucleic acid molecule to be tested and the primer, and the duplex is fixed on the support; (2) the primer in the duplex is used as a first initial growth nucleic acid strand, Under the catalysis of polymerase, one or two of the first to fifth nucleotide analogs are incorporated into the 3 ′ end of the initial growing nucleic acid strand, so that Only one first new nucleotide is extended at the 3 ′ end to form a first product duplex; (3) the polymerase in the reaction system (1) and (2) is removed and the unreacted first to fifth nuclei Nucleotide analogs; (4) judging the first nucleotide at the 3 'end of the nucleic acid molecule to be tested based on the fluorescence signal and phosphorescence signal of the first product duplex; wherein the first to fifth nucleotides The analog has a base complementary pairing ability, and the ribose or deoxyribose at the 3 ′ position of the first to fifth nucleotide analogs Hydroxyl group is protected by a protecting group, the protecting group is a polymerase reaction blocking group, the first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth The nucleotide analog has a different base from the fifth nucleotide analog, the first nucleotide analog has the same base as the third nucleotide analog, and the first A nucleotide analog has a different base from the second nucleotide analog, and the first nucleotide analog and the second nucleotide analog are different from the fourth nucleotide analog Have different bases, and the third nucleotide analog and the fourth nucleotide analog have different bases, and the first and second nucleotide analogs each independently carry a fluorescent group Group, does not carry a 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 phosphorescence Group. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors devised a new method for sequencing nucleic acids, using the optical signals of the first to fifth nucleotide analogs (while There are phosphorescence and fluorescence, only phosphorescence and no fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to identify the corresponding bases (A, T / U, C and G) in the nucleic acid molecule to be sequenced. With the sequencing method according to the 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 cost of sequencing is reduced, and the sequencing results are accurate.

In a second aspect of the invention, the invention proposes a nucleotide analog. According to an embodiment of the present invention, the nucleotide analog has a structural formula represented by formula (I),

Among them, 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 resistance R ₁ is -OH or -H, and P ₁ is H or a phosphate group. The inventors first proposed and designed a nucleotide analog with a phosphorescent group. The nucleotide analog with a phosphorescent group could be used alone in DNA and / or RNA sequencing, replacing the two-color fluorescence sequencing in the prior art. Method, monochromatic fluorescence sequencing and other fluorescent dye-based sequencing methods that use fluorescent analogs that carry fluorescent groups, and use the optical properties of phosphorescent group-containing nucleotide analogs and fluorescent group-based analogs The difference can realize a sequencing method that uses the optical properties of nucleotide analogs to distinguish bases for the first time in this application. The nucleotide analog according to the embodiment of the present invention carries a phosphorescent group, and is a new nucleotide analog that can be used in DNA and / or RNA sequencing.

In a third aspect of the invention, the invention proposes a nucleotide analog mixture. According to an embodiment of the present invention, the nucleotide analog mixture includes the aforementioned nucleotide analog. The inventors first proposed a nucleotide analog mixture containing a nucleotide analog carrying a phosphorescent group, and the phosphorescent optical signal could be generated by using the nucleotide analog carrying a phosphorescent group in the nucleotide analog mixture To achieve 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 present invention, the nucleotide analog mixture includes: a first nucleotide analog and a second nucleotide analog, each of which has a structural formula shown by Formula II,

Among them, Base ₂ represents adenine, guanine, cytosine, thymine or uracil; D ₂ represents a fluorophore; C ₂ represents a cleavable bond or an optionally substituted cleavable group; B ₂ represents a polymerase reaction resistance R ₂ is -OH or -H; P ₂ represents H or a phosphate group;

The third nucleotide analog and the fourth nucleotide analog, each of which has a structural formula shown in Formula I,

Among them, 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 resistance R ₁ is -OH or -H; P ₁ is H or phosphate group;

A fifth nucleotide analog, which has the structural formula shown in Formula III,

Among them, Base ₃ represents 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;

The first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth nucleotide analog have different bases from 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 first nucleotide analog Tetranucleotide analogs have different bases. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors designed a mixture of nucleotide analogs and used the first to fifth nucleotide analogs for nucleic acid sequencing. Detect different optical signals (with both phosphorescence and fluorescence, only phosphorescence and no fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to quickly and accurately identify the type of the corresponding base on the nucleic acid chain, and use the nuclear according to the embodiment of the present invention For the sequencing of the nucleotide analog mixture, only one excitation light and one filtering device are needed, and no compound reaction is required between the two detection signals, thereby simplifying the sequencing steps, reducing sequencing costs, and accurate sequencing results.

In a fifth aspect of the invention, the invention provides a kit. According to an embodiment of the present invention, the kit includes: the aforementioned nucleotide analogue, or the aforementioned nucleotide analogue mixture. The inventors first proposed and designed a nucleotide analog carrying a phosphorescent group. The nucleotide analog carrying a phosphorescent group can be used alone in DNA and / RNA sequencing, replacing the two-color fluorescence sequencing method in the prior art, Monochrome fluorescence sequencing and other fluorescent-sequence-containing nucleotide analogs are used in sequencing methods such as fluorescent dyes. At the same time, the differences in the optical properties of the nucleotide analogs carrying phosphorescent groups and the analogs of fluorescent groups can be used to realize the sequencing method that uses the optical properties of the compounds to distinguish bases for the first time in this application. The nucleotide analog mixture, and the aforementioned nucleotide analog mixture is applied to the preparation of the kit. The kit according to the embodiment of the present invention has low cost, simple operation, and can quickly and accurately detect the type of bases in a nucleic acid molecule.

In a sixth aspect of the invention, the invention provides a method for performing a polymerase reaction. According to an embodiment of the present invention, the method includes: (1) placing a mixture containing a single-stranded template, a primer, the aforementioned nucleotide analog mixture, and a polymerase under conditions suitable for primer extension, wherein The primer matches a portion of the single-stranded template so that only one first new nucleotide is extended at the 3 'end of the primer. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors designed a mixture of nucleotide analogs and used the first to fifth nucleotide analogs for nucleic acid sequencing. By detecting different optical signals (with both phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence), the type of the corresponding base in the nucleic acid chain can be accurately identified. According to the method of the embodiment of the present invention, nucleotide analogues can be accurately paired with nucleic acid strands, thereby facilitating nucleic acid sequencing quickly and accurately.

In a seventh aspect of the present invention, the present invention provides a method for determining a nucleic acid sequence. According to an embodiment of the present invention, the method includes: performing a controllable chain polymerase reaction on the nucleic acid sequence to be sequenced by using the method described above. According to the method of the embodiment of the present invention, only one excitation light and one lens of a filtering device are required during the sequencing process, and no chemical reaction is required between two photographs, simplifying the sequencing steps, reducing sequencing time, and sequencing. Cost and accurate sequencing results.

In an eighth aspect of the present invention, the present invention provides a sequencer. According to an embodiment of the present invention, the sequencer includes: a casing; a chain polymerase reaction region, the chain polymerase reaction region is disposed in the casing; an excitation light emitter, and the laser emitter is suitable Emitting a predetermined wavelength of excitation light to the chain polymerase reaction area; and a signal acquisition device, the signal acquisition device is adapted to collect fluorescence signals and phosphorescence signals in the chain polymerase reaction area. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors designed a mixture of nucleotide analogs and used the first to fifth nucleotide analogs for nucleic acid sequencing. By detecting different optical signals (with both phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to identify the type of the corresponding base of the nucleic acid chain, and based on the above sequencing method, the inventor proposed A new sequencer requires only an excitation light and a lens of a filtering device, and does not require a chemical reaction between two pictures, simplifying the hardware conditions and sequencing steps of the sequencer and reducing Instrument cost and reagent cost. The sequencer according to the embodiment of the present invention has low cost, simple sequencing process, short sequencing time, and accurate sequencing results, which is beneficial to the miniaturization and development of the sequencer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a sequencer according to an embodiment of the present invention;

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, where the left side is a fluorescent channel image and the right side is a phosphorescent channel image.

Reference signs:

Sequencer 1000, housing 100, chain polymerase reaction area 11, laser transmitter 12, signal acquisition device 13, and controller 14.

detailed description

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention, but should not be construed as limiting the present invention.

Method for sequencing nucleic acid molecules

In a first aspect of the invention, the invention provides a method for sequencing a nucleic acid molecule. According to an embodiment of the present invention, the method includes: (1) annealing a nucleic acid molecule to be tested with a primer so as to form an initial duplex, the nucleic acid molecule or primer to be tested is previously fixed on a support, so The duplex is composed of the nucleic acid molecule to be tested and the primer, and the duplex is fixed on the support; (2) the primer in the duplex is used as a first initial growth nucleic acid strand, Under the catalysis of polymerase, one or two of the first to fifth nucleotide analogs are incorporated into the 3 ′ end of the initial growing nucleic acid strand, so that Only one first new nucleotide is extended at the 3 ′ end to form a first product duplex; (3) the polymerase in the reaction system (1) and (2) is removed and the unreacted first to fifth nuclei Nucleotide analogs; (4) judging the first nucleotide at the 3 'end of the nucleic acid molecule to be tested based on the fluorescence signal and phosphorescence signal of the first product duplex; wherein the first to fifth nucleotides The analog has a base complementary pairing ability, and the ribose or deoxyribose at the 3 ′ position of the first to fifth nucleotide analogs Hydroxyl group is protected by a protecting group, the protecting group is a polymerase reaction blocking group, the first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth The nucleotide analog has a different base from the fifth nucleotide analog, the first nucleotide analog has the same base as the third nucleotide analog, and the first A nucleotide analog has a different base from the second nucleotide analog, and the first nucleotide analog and the second nucleotide analog are different from the fourth nucleotide analog Have different bases, the third nucleotide analog and the fourth nucleotide analog have different bases, and the first and second nucleotide analogs each independently carry a fluorescent group , Does not carry a 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 group. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors devised a new method for sequencing nucleic acids, using the optical signals of the first to fifth nucleotide analogs (while There are phosphorescence and fluorescence, only phosphorescence and no fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to identify the corresponding bases (A, T / U, C and G) in the nucleic acid molecule to be sequenced. With the sequencing method according to the 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 cost of sequencing is reduced, and the sequencing results are accurate.

According to an embodiment of the present invention, the method further comprises: (5) cleavage the first product duplex in a reaction system containing a solution phase and a solid phase, so as to remove the nucleotide analog Protecting group and / or light signal group at the 3 'position of ribose or deoxyribose, (6) removing the solution phase of the reaction system in step (5), (7) in step (6) in the reaction system The cleavage treatment product is a second initial growth nucleic acid chain, and one or two of the first to fifth nucleotide analogs are incorporated into 3 'of the initial growth nucleic acid chain under the catalysis of a 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) repeat steps (3) and (4) to determine whether to wait The second nucleotide sequence at the 3 'end of the nucleic acid molecule is measured. By cutting the protective group at the 3 'position on the nucleotide analog to ensure the next polymer chain reaction, and by cutting the luminescent group (fluorescent or phosphorescent group) on the nucleotide analog ) In order to better detect the optical signal carried by the nucleotide analogue on the next round of polymerase chain reaction amplification. It should be noted that the solution phase referred to in the present invention refers to a reaction liquid in which a reaction occurs, and the solid phase refers to a support on which a template is fixed.

According to an embodiment of the present invention, the extended first new nucleotide is a first or second nucleotide analog, and the cleaved light signal group is a fluorescent group.

According to an embodiment of the present invention, the extended first new nucleotide is a third and fourth nucleotide analog, and the cleaved light signal group is a phosphorescent group.

According to an embodiment of the present invention, the extended first new nucleotide is a fifth nucleotide analog, and only the protective group at the 3 ′ position of the ribose or deoxyribose in the protective group is cleaved.

According to an embodiment of the present invention, the method further includes the following step (9): The method further includes the following step (9): (9) repeating steps (5) to (8) one or more times so that Determine the nucleotide sequence of the nucleic acid molecule to be tested. Repeat steps (5) to (8) one or more times until the entire sequence of the nucleic acid molecule to be sequenced is measured.

According to an embodiment of the present invention, the simultaneous presence of 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, and the fluorescent signal exists, but not 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 second nucleotide analog, and the fluorescent signal does not exist, but the presence of the phosphorescent signal indicates that the The base corresponding to the sequenced nucleic acid molecule is the base corresponding to the fourth nucleotide analog. Therefore, the bases corresponding to the nucleic acid molecules to be sequenced can be determined quickly and accurately through different optical signals.

According to an embodiment of the present invention, the nucleic acid molecule to be tested is DNA, and the nucleic acid molecule to be tested is subjected to denaturation treatment in advance to obtain a single-stranded nucleic acid molecule to be tested.

Nucleotide analogs

It should be noted that the phosphorescent group is not particularly limited, as long as the phosphorescent group satisfies the condition that it can emit a controllable detection phosphorescent signal under specific excitation light conditions. For example, the phosphorescent group may have the following structure:

Wherein, R ₄ is H, F, Cl, Br, I, CN, NO ₂ , C _1-6 alkyl, C _1-6 haloalkyl, and C _1-6 alkoxy. The above groups are capable of emitting phosphorescence under the action of specific excitation light.

According to an embodiment of the present invention, the phosphate group is a monophosphate group, a bisphosphate group, a triphosphate group, or a polyphosphate group.

It should 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 cleavable group commonly used in the prior art including the following groups -SS-, CH ₂ CH = CH ₂ , -CH ₂ N ₃ and the like.

According to an embodiment of the present invention, the C ₁ further includes 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 at the polymerase reaction site in order to prevent the polymerase chain reaction Or, the reaction is performed at the above-mentioned sites during the sequencing process to affect the sequencing results.

For example, the C ₁ includes, but is not limited to, the following structure:

Here, n1 to n17 are each independently an integer between 0 and 7.

It should be noted that the polymerase reaction blocking group is not particularly limited, as long as it can meet the requirement to block the site for the polymerase reaction under a specific condition, and can be removed under another specific condition for the polymerase reaction Just fine. For example, the polymerase reaction blocking group may be a common polymerase reaction blocking group including the following groups: -SS-, CH ₂ CH = CH ₂ , and -CH ₂ N ₃ . The above-mentioned groups can be controlled to amplify only one new nucleotide at a time during the polymerase chain reaction or the sequencing process, and then detect the optical signal, and then excise it for the next round of amplification.

According to an embodiment of the present invention, the structure shown by formula (I) is one of the following structures:

Nucleotide Analog Mixture

The first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth nucleotide analog have different bases from 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 first nucleotide analog Tetranucleotide analogs have different bases. Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors designed a mixture of nucleotide analogs and used the first to fifth nucleotide analogs for nucleic acid sequencing. Detect different optical signals (with both phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to quickly and accurately identify the type of the corresponding base on the nucleic acid chain, and use the nuclear For the sequencing of the nucleotide analog mixture, only one excitation light and one filtering device are needed, and no compound reaction is required between the two detection signals, thereby simplifying the sequencing steps, reducing sequencing costs, and accurate sequencing results.

According to an embodiment of the present invention, the C ₁ and / or C ₂ further include 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 at the polymerase reaction site in order to prevent the polymerase chain reaction Or, the reaction is performed at the above-mentioned sites during the sequencing process to affect the sequencing results.

For example, the C ₁ and / or C ₂ include but are not limited to the following structures:

Here, n1 to n17 are each independently an integer between 0 and 7.

It should be noted that the polymerase reaction blocking group is not particularly limited, as long as it can meet the requirement to block the site for the polymerase reaction under a specific condition, and can be removed under another specific condition for the polymerase reaction to proceed. Just fine. For example, the polymerase reaction blocking group may be a common polymerase reaction blocking group including the following groups: -SS-, CH ₂ CH = CH ₂ , and -CH ₂ N ₃ . The above-mentioned groups can be controlled to amplify only one new nucleotide at a time during the polymerase chain reaction or the sequencing process, and then detect the optical signal, and then excise it for the next round of amplification.

It should be noted that the fluorescent group is not particularly limited, as long as the fluorescent group satisfies the condition that it can emit a controllable and detectable fluorescent signal under specific excitation light conditions. For example, D ₂ includes but is not limited to the following structures:

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):

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

According to an embodiment of the present invention, the kit further includes: a cutting reagent, the cutting reagent may act on a cleavable group or a cleavable bond. The aforementioned cleavage reagent can cut the cleavable group or cleavable bond in the aforementioned nucleotide analog or nucleotide analog mixture, thereby removing the polymerase reaction blocking group and exposing the polymerase reaction. Site for the next new base amplification.

It should be noted that the cleavage reagent is not particularly limited, as long as it can satisfy the cleavable bond or cleavable group in the nucleotide analog, and the nucleic acid molecule to be sequenced and the polymerase chain reaction will not cause substantial influence. For example, TCEP / THPP can be used as a cutting reagent to efficiently cut disulfide bonds, and organic phosphides can be used as a cutting reagent to efficiently cut azide groups.

Method of polymerase reaction

According to an embodiment of the present invention, the single-stranded template or primer is fixed on a solid-phase support.

According to an embodiment of the present invention, the single-stranded template or primer is fixed on a chip.

According to an embodiment of the present invention, the method further comprises: (2) cutting off the polymerase reaction blocking group of the new nucleotide, and returning to step (1) so that the first new nucleoside The 3 'end of the acid continues to extend by only a second new base. Facilitating the above method, nucleic acid molecules can be accurately and quickly sequenced.

According to an embodiment of the present invention, after step (1), the method further includes: (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), the method further includes: removing a reactant in the system after extending the first new nucleotide. By removing the reactants 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), the method further includes: (1-2) determining the first new base and the single-stranded template based on at least one of the fluorescent signal and the phosphorescent signal. And a type corresponding to at least one base at a position corresponding to the first new base.

According to an embodiment of the present invention, the simultaneous existence of the fluorescent signal and the phosphorescent signal indicates that the base of the first new nucleotide is a base corresponding to the first nucleotide analog and the third nucleotide analog. Base, neither the fluorescent signal nor the phosphorescent signal indicates that the base of the first new nucleotide is the base corresponding to the fifth nucleotide analog, and the fluorescent signal is present but not The presence of the phosphorescent signal indicates that the base of the first new nucleotide is the base corresponding to the second nucleotide analog, and the fluorescent signal does not exist, but the presence of the phosphorescent signal indicates that the The base of a new nucleotide is the base corresponding to the fourth nucleotide analog. Therefore, according to the difference in the optical signal of the base of the first new nucleotide, the type of the base at the corresponding position in the nucleic acid molecule to be sequenced can be accurately identified.

According to the embodiment of the present invention, in step (1-2), an excitation wavelength of 400 to 480 nm and a signal acquisition filter of 500 to 570 nm are used. The inventor found that based on the above excitation wavelength and signal acquisition filter, fluorescence signals and phosphorescence signals can be detected successively, and only one excitation light and one signal acquisition filter are needed to meet the detection requirements. At the same time, two signals No chemical reaction is required during the acquisition. As a result, the time and cost of sequencing are saved.

According to an embodiment of the present invention, the fluorescent signal is collected during a time period when the excitation light is turned on, and the phosphorescent signal is collected between 0.5 to 100 milliseconds after the excitation light is turned off. By using the method of the embodiment of the present invention to perform a polymerase reaction, no chemical reaction is required between two signal acquisitions, and the time interval between the two signal acquisitions is short. As a result, the time and cost of sequencing are saved.

Method for determining nucleic acid sequence

Sequencer

In a sixth aspect of the invention, the invention provides a sequencer. 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 is disposed in the casing 100;

The excitation light emitter 12 is adapted to emit excitation light of a predetermined wavelength to the chain polymerase reaction region 11. According to a specific embodiment of the present invention, the predetermined wavelength is 400-480 nm.

And a signal acquisition device 13, the signal acquisition device 13 is adapted to acquire a fluorescent signal and a phosphorescent signal in the chain polymerase reaction region 11.

According to another specific embodiment of the present invention, referring to FIG. 2, the sequencer 1000 further includes a controller 14, which is respectively connected to the signal acquisition device 13 and the laser transmitter 12 for controlling The excitation light emitter 12 is turned on and off, and the signal acquisition device 13 is controlled to switch between acquiring a fluorescent signal and the phosphorescent signal. According to still another specific embodiment of the present invention, the controller 14 is adapted to control the signal acquisition device 13 to collect a fluorescent signal during the activation process of the excitation light emitter 12, and to turn off the excitation light emitter 12 The phosphorescent signal is then acquired within a predetermined time range. According to another specific embodiment of the present invention, the predetermined time is 0.5 to 100 milliseconds.

Based on this principle, the inventors first proposed the use of differences in the optical properties of compounds to distinguish bases. Based on this principle, the inventors designed a mixture of nucleotide analogs and used the first to fifth nucleotide analogs for nucleic acid sequencing. By detecting different optical signals (with both phosphorescence and fluorescence, only phosphorescence without fluorescence, only fluorescence without phosphorescence, no phosphorescence and fluorescence) to identify the type of the corresponding base of the nucleic acid chain, and based on the above sequencing method, the inventor proposed A new sequencer requires only an excitation light and a lens of a filtering device, and does not require a chemical reaction between two pictures, simplifying the hardware conditions and sequencing steps of the sequencer and reducing Instrument cost and reagent cost. The sequencer according to the embodiment of the present invention has low cost, simple sequencing process, short sequencing time, and accurate sequencing results, which is beneficial to the miniaturization and development of the sequencer.

Preparation

The compounds represented by formula (II) and formula (III) in the nucleotide analog mixtures involved in this application are common nucleotide analogs in the prior art, and can be synthesized by referring to methods in the prior art, such as Reference can be made to the methods in WO 2017 / 058953A1, WO 2017 / 087887A1, US 2017 / 0130051A1 and other patents for synthesis.

The compound represented by formula (I) in the nucleotide analog mixture involved in the present application is a nucleotide analog proposed by the inventor for the first time, and its synthesis method can also be synthesized with reference to the methods in the prior art, the difference is that It needs to convert the fluorescent group into the phosphorescent group for synthesis in the synthesis process. For the synthesis of the phosphorescent group, reference may be made to the synthesis method of the related phosphorescent group in the prior art, for example, the method in Fraser, C.L., NATURE MATERIALS, 2009, 08,747-751 (DOI: 10.1038 / NMAT2509) can be used.

For example, using the methods in Fraser, C.L., NATURE MATERIALS, 2009, 08,747-751 (DOI: 10.1038 / NMAT2509), the following raw materials can be obtained:

n can choose any integer (including 0) as required,

Use the methods in WO2017 / 058953A1, WO2017 / 087887A1, US 2017 / 0130051A1 and other patents to obtain the raw material 2 with the fluorescent group to be connected (in the present invention, it can also be considered as the raw material 2 with the phosphorescent group to be connected);

Then, the above-mentioned raw material 1 and the raw material 2 to which the phosphorescent group is to be attached are subjected to a classical chemical reaction such as a condensation reaction, a substitution reaction, or an addition reaction under suitable conditions for synthesis, so as to obtain a nucleotide analog carrying a 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:

碱基类型Base type	荧光通道Fluorescence channel	磷光通道Phosphorescent channel
AA	发光Glow	不发光No light
GG	不发光No light	不发光No light
CC	不发光No light	发光Glow
TT	发光Glow	发光Glow

Among them, T bases use a mixture of phosphorescent and fluorescently modified dTTP.

In order to verify the base discrimination based on this principle, under the fluorescence microscope, several DNAs with different sequences were fixed on the glass chip by a spotter. The diameter of each spot is 50 micrometers. The excitation wavelength of the fluorescence microscope is selected in the range of 400-480 nanometers, and the acquisition signal filters of fluorescence and phosphorescence are in the range of 500-570 nanometers. The fluorescence signal is collected during the period when the excitation light is on, and the phosphorescence signal is collected between 0.5 ms and 100 ms after the excitation light is turned off.

In the verification experiment, the reagents used in the BGISEQ-500 reagent and various buffers. The fluorescently modified probes of sequencing reagents were changed to the above-mentioned five fluorescent and phosphorescently modified reversible blocking nucleotides.

First purchased the DNA array on the silicon chip (Boao Biochip Customization Service), using four DNAs of the same sequence, the four sequences at each position corresponding to the measured area have different bases, and contain four bases The four sequences are shown in Table 2 below.

Table 2:

After the DNA is immobilized, sequencing primers (as in Table 2 above) are added for partially complementary sequences. After adding a DNA polymerase mixture containing the above five nucleotides to a silicon wafer, under the PCR extension conditions, the 3 ′ end of each DNA primer is polymerized with the corresponding base, and then the unreacted nucleotides are washed away. Phosphate buffered solution containing vitamin C was added, and images were collected under a fluorescence microscope. Phosphorescent signal images were obtained by setting the signal collection time of the microscope.

The first cycle first obtained Figure 3, the image on the left is the image of the fluorescence channel, and the image on the right is the image of the phosphorescence channel. The leftmost two columns are the mixed DNA localization regions, so the 6 points have signals in both channels. Ten columns are a single type of DNA sequence points, with a total of 6 × 8 points.

After 5 cycles of sequencing, the sequence of the above 48 points was determined respectively;

4a, 4e, 4f, 5a, 5e, 6a, 6c, 7a, 8b, 8c, 8f, 9b, 9c, 9e, 10c, 10d, and 10e are sequences 1.

3b, 3c, 3e, 4c, 6b, 7b, 9a and 10b are sequences 2.

3a, 4d, 5b, 5c, 6f, 7d, 7f, 8a, 9f and 10a are sequences 3.

3d, 3f, 4b, 5d, 5f, 6d, 6e, 7c, 7e, 8d, 8e, 9d, and 10f are sequences 4.

The above sequence is exactly the same as the sequence designed by the custom chip, and the short sequencing read length is 100% accurate.

In the description of this specification, the description with reference to the terms “one embodiment”, “some embodiments”, “examples”, “specific examples”, or “some examples” and the like means specific features described in conjunction with the embodiments or examples , Structures, materials, or features are included in at least one embodiment or example of the invention. In this specification, the schematic expressions 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. In addition, without any contradiction, those skilled in the art may combine and combine different embodiments or examples and features of the different embodiments or examples described in this specification.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limitations on the present invention. Those skilled in the art can interpret the above within the scope of the present invention. Embodiments are subject to change, modification, substitution, and modification.

Claims

A method for sequencing a nucleic acid molecule, characterized in that:

(1) An annealing reaction is performed between a nucleic acid molecule to be tested and a primer so as to form an initial duplex. The nucleic acid molecule or primer to be tested is fixed on a support in advance, and the duplex is composed of the nucleic acid molecule to be tested and The primer is configured, and the duplex is fixed on the support;

(2) using the primers in the duplex as the first initial growth nucleic acid strand, and catalyzing by polymerase, incorporating one or two of the first to fifth nucleotide analogs into the duplex The 3 'end of the initial growing nucleic acid strand, so that only one first new nucleotide is extended at the 3' end of the initial growing nucleic acid strand, forming a first product duplex;

(3) removing the polymerase and the unreacted first to fifth nucleotide analogs in the reaction system of steps (1) and (2);

(4) judging the first nucleotide at the 3 ′ end of the nucleic acid molecule to be tested based on the fluorescence signal and phosphorescence signal of the first product duplex;

Wherein, the first to fifth nucleotide analogs have a base complementary pairing ability, and 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. The protective group is a polymerase reaction blocking group,

The first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth nucleotide analog have different bases from the fifth nucleotide analog,

The first nucleotide analog has the same base as the third nucleotide analog,

The first nucleotide analog has a different base from the second nucleotide analog,

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 not a phosphorescent group,

The third and fourth nucleotide analogs each independently carry a phosphorescent group and no fluorescent group,

The fifth nucleotide analog does not carry a fluorescent group and a phosphorescent group.
The method according to claim 1, further comprising:

(5) The first product duplex is subjected to a cleavage treatment in a reaction system containing a solution phase and a solid phase, so as to remove the protecting group at the 3 ′ position of the ribose or deoxyribose in the nucleotide analog. And / or optical signalling groups,

(6) removing the solution phase of the reaction system in step (5),

(7) Taking the cleavage treatment product in the reaction system of step (6) as the second initial growth nucleic acid chain, and catalyzing by polymerase, one or two of the first to fifth nucleotide analogs are combined Entering the 3 ′ end of the initial growth nucleic acid strand so that only a second new nucleotide is extended at the 3 ′ end of the second initial growth nucleic acid strand to form a second product duplex;

(8) Repeat steps (3) and (4) to determine the second nucleotide sequence at the 3 ′ end of the nucleic acid molecule to be tested.
The method according to claim 2, wherein the extended first new nucleotide is a first or second nucleotide analog, and the cleaved light signal group is a fluorescent group;

Optionally, the extended first new nucleotide is a third or fourth nucleotide analog, and the cleaved light signal group is a phosphorescent group;

Optionally, the extended first new nucleotide is a fifth nucleotide analog, which only cleaves the protecting group at the 3 'position of the ribose or deoxyribose in the protecting group.
The method according to claim 2, further comprising the following step (9):

(9) Repeat steps (5) to (8) one or more times to determine the nucleotide sequence of the nucleic acid molecule to be tested.
The method according to claim 1, wherein the simultaneous presence of the fluorescent signal and the phosphorescent signal indicates that the base corresponding to the nucleic acid molecule to be sequenced is the first nucleotide analog and the third nucleus Bases corresponding to the nucleotides,

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;

Optionally, the test nucleic acid molecule is DNA, and the test nucleic acid molecule is previously subjected to denaturation treatment in order to obtain a single-stranded test nucleic acid molecule.
A nucleotide analogue characterized in that it has a structural formula represented by formula (I),

among them,

Base 1 means 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 nucleotide analog mixture, comprising the nucleotide analog according to claim 5.
A mixture of nucleotide analogs, comprising:

The first nucleotide analog and the second nucleotide analog, each independently having a structural formula shown by Formula II,
among them,

Base 2 means 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;

The third nucleotide analog and the fourth nucleotide analog, each of which has a structural formula shown in Formula I,

among them,

Base 1 means 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, which has the structural formula shown in Formula III,

among them,

Base 3 means

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;

The first nucleotide analog, the second nucleotide analog, the third nucleotide analog, and the fourth nucleotide analog have different bases from the fifth nucleotide analog. ;

The first nucleotide analog has the same base as the third nucleotide analog;

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 has a different base from the fourth nucleotide analog.
The nucleotide analogue according to claim 6 or the nucleotide analog mixture according to claim 7 or the nucleotide analog mixture according to claim 8, wherein the structure represented by formula (I) is Structure of one of the following:
The nucleotide analog mixture according to 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 includes:

The nucleotide analogue of claim 6; or

The nucleotide analog mixture of claim 7; or

The nucleotide analog mixture according to claim 8.
The kit according to claim 11, further comprising:

Cleavage reagents that can act on cleavable groups or cleavable bonds.
A method for performing a polymerase reaction, comprising:

(1) placing a mixture containing a single-stranded template, a primer, the nucleotide analog mixture according to claim 8 and a polymerase under conditions suitable for primer extension,

Wherein, the primer matches a part of the single-stranded template so that only one first new nucleotide is extended at the 3 ′ end of the primer.
The method according to claim 13, wherein the single-stranded template or primer is immobilized on a solid support.
The method according to claim 14, wherein the single-stranded template or primer is fixed on a chip.
The method according to claim 13, further comprising:

(2) the polymerase reaction blocking group of the first new nucleotide is excised and returned to step (1) so as to continue at the 3 ′ end of the ribose or deoxyribose of the first new nucleotide Extend only one second new nucleotide.
The method according to claim 13, wherein after step (1), further comprising:

(1-1) The fluorescence signal and the phosphorescence signal of the first new base are detected separately.
The method according to claim 17, further comprising: before step (1-1), removing reactants in the system after extending the first new nucleotide.
The method according to claim 17, further comprising, after step (1-1):

(1-2) determining, based on at least one of the fluorescent signal and the phosphorescent signal, at least one of the first new base and at least one base at a position corresponding to the first new base on the single-stranded template; Types of.
The method according to claim 19, wherein the simultaneous presence of the fluorescent signal and the phosphorescent signal indicates that the base of the first new nucleotide is the first nucleotide analog and the third nucleotide Bases for analogs,

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 absence of the fluorescent signal, but the presence of the phosphorescent signal indicates that the first new nucleotide is a base corresponding to the fourth nucleotide analog.
The method according to claim 19, wherein in step (1-2), an excitation wavelength of 400 to 480 nm and a signal acquisition filter of 500 to 570 nm are used.
The method according to claim 19, wherein the fluorescent signal is collected during a time period when the excitation light is turned on, and the phosphorescent signal is collected between 0.5 to 100 milliseconds after the excitation light is turned off.
A method for determining a nucleic acid sequence, comprising: performing a controllable-chain polymerase reaction on a nucleic acid sequence to be tested by using the method according to any one of claims 13 to 22.
A sequencer, comprising:

case;

A chain polymerase reaction region, the chain polymerase reaction region is disposed in the shell;

An excitation light emitter adapted to emit excitation light of a predetermined wavelength to the chain polymerase reaction region; and

A signal acquisition device adapted to acquire a fluorescent signal and a phosphorescent signal in the reaction region of the chain polymerase.
The sequencer according to claim 24, wherein the signal acquisition device comprises:

Cameras; and

A filter, the filter range of which is 500-570 nm.
The sequencer according to claim 24, wherein the sequencer further comprises a controller, and the controller is respectively connected to the signal acquisition device and the laser transmitter for controlling the excitation light emission Turning on and off the transmitter, and controlling the signal acquisition device to switch between collecting the fluorescent signal and the phosphorescence signal, the controller is adapted to control the signal acquisition device during the activation of the excitation light emitter A fluorescent signal is collected, and the phosphorescent signal is collected within a predetermined time range after the excitation light emitter is turned off.