阅读说明：本技术 一种优化的扩增目标核酸的方法及应用 (Optimized method for amplifying target nucleic acid and application ) 是由 杨国华 郭志伟 林国旻 车彬 余佳佳 李�杰 于 2020-05-11 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种优化的扩增目标核酸的方法,包括：使结合了引物的模板和至少部分经标记分子修饰的dNTP,以及具有3’-5’外切酶活性的高保真聚合酶DNA聚合酶接触得到扩增产物,得到得率和标记分子掺入率均理想的纯化产物。本发明还公开了用于所述方法的具有高掺入率的dNTP-DNA聚合酶复合体系、经改造的DNA聚合酶、经标记分子修饰的dNTP。本发明提供的方法及复合体系、经改造的DNA聚合酶、经标记分子修饰的dNTP,对不定长度的单链扩增实现了显著的技术进步,具有广泛的应用前景和显著的经济价值。(The invention relates to the technical field of biology, and discloses an optimized method for amplifying target nucleic acid, which comprises the following steps: the template combined with the primer is contacted with dNTP at least partially modified by the labeled molecule and high-fidelity polymerase DNA polymerase with 3'-5' exonuclease activity to obtain an amplification product, and a purified product with ideal yield and labeled molecule incorporation rate is obtained. The invention also discloses a dNTP-DNA polymerase complex system with high incorporation rate, a modified DNA polymerase and a dNTP modified by a marker molecule for the method. The method and the composite system provided by the invention, the modified DNA polymerase and the dNTP modified by the marker molecule realize obvious technical progress on the amplification of single strands with indefinite length, and have wide application prospect and obvious economic value.)

1. An optimized method for amplifying a target nucleic acid comprising the steps of:

in a reaction system, target nucleic acid molecules are taken as templates, primers are added, a dNTP-DNA polymerase complex system with high doping rate is obtained, and amplification products of the target nucleic acids are obtained through amplification reaction;

it is characterized in that the preparation method is characterized in that,

the high-incorporation-rate dNTP-DNA polymerase complex system comprises dNTP and DNA polymerase;

the dNTP is a dNTP at least partially modified by a marker molecule, and when the DNA polymerase contains site-directed mutation of an N-terminal domain, the modification group in the dNTP modified by the marker molecule contains or does not contain a double-bond or triple-bond structure for preventing the modification group molecule from rotating in the first 3C atoms connected with the dNTP base group; when the DNA polymerase does not contain site-directed mutation of the N-terminal domain, the first 3C atoms of the dNTP modified by the marker molecule, to which the modification group is connected with the base group of the dNTP, do not contain a double-bond or triple-bond structure for preventing the modification group molecule from rotating;

the DNA polymerase is a high fidelity polymerase with 3'-5' exonuclease activity.

2. The method of claim 1,

the DNA polymerase is one or more of Pfu, Deep Vent, KOD; and/or the presence of a gas in the gas,

the DNA polymerase is obtained by modifying one or more of Pfu, Deep Vent and KOD;

the dNTP modified by the marker molecule is one or more of dATP, dCTP and dGTP; when the DNA polymerase contains the V93Q mutation, the modifying group in the dNTP modified by the marker molecule contains or does not contain a double-bond or triple-bond structure for preventing the modifying group molecule from rotating in the first 3C atoms connected with the dNTP base group; when the DNA polymerase does not contain the V93Q mutation, the first 3C atoms of the dNTP modified by the marker molecule, to which the modification group is connected with the base group of the dNTP, do not contain a double-bond or triple-bond structure for preventing the modification group molecule from rotating;

the marker molecule is biotin;

the amplification is single-stranded linear amplification.

3. The method of claim 2,

the biotin-containing modification group binds to the dATP at the site of base N6 or 7-Deaza;

preferably, when the site where the biotin-containing modifying group binds to the dATP is N6 of the base, the biotin-modified dATP is biotin-7-dATP or biotin-14-dATP, and the structural formula is shown below;

when the site of the biotin-containing modification group combined with the dATP is 7-Deaza of a base, the biotin-modified dATP is biotin-11-dATP, and the structural formula is shown as follows;

and/or the site where the biotin-containing modification group binds to dCTP is N4 or C5 of base;

preferably, when the site at which the biotin-containing modifying group binds to dCTP is N4 of base, the biotin-modified dCTP is biotin-14-dCTP, the structural formula of which is shown below;

when the site at which the biotin-containing modifying group binds to dCTP is C5 of a base, the biotin-modified dCTP is biotin-11-dCTP or biotin-16-dCTP, and the structural formula is shown below;

and/or, the biotin-containing modifying group binds to the dGTP at a site that is the base 7-Deaza;

preferably, the biotin-modified dGTP is biotin-11-dGTP, and the structural formula is shown as follows;

4. the method of claim 2,

the modification is that a double-stranded DNA binding domain is added on the DNA polymerase;

preferably, the alteration is the addition of an Sso7d domain at the C-terminus of the DNA polymerase;

more preferably, the modification is that an Sso7d domain is added to the C terminal of Pfu polymerase to form a caudate-added body 1; or, the modification is that a Sso7d structural domain is added to the C end of KOD polymerase to form a plus-tail body 2; alternatively, the modification is to add an Sso7d domain to the C-terminus after Pfu and DeepVent polymerases are chimeric to form chimera 1.

5. The method of claim 4,

the modification also comprises carrying out strand displacement on the episome 2 and the chimera 1 to form displacers 1 and 2;

wherein the replacement body 1 is formed by fusing the C end of the chimera 1 and the N end of the tail body 2,

the displacement body 2 is formed by fusing the N end of the chimera 1 and the C end of the tail body 2;

preferably, the site of strand displacement is between the 3'-5' exonuclease active region and the polymerase active region;

more preferably, the site of strand displacement is between the 300 th and 360 th amino acids of the N-terminus;

more preferably, the site of strand displacement is between amino acids 326-327 of the N-terminus;

more preferably, the modification further comprises the step of setting a point mutation near the N-terminus of the substituent 2 to form mutant 1;

more preferably, the site of the point mutation is between 1-100 amino acids from the N-terminus;

more preferably, the position of the point mutation is V93Q.

6. The method of claim 2,

the dNTP is an equimolar mixture of two or more of dATP, dCTP, dGTP and dUTP, and the mixing molar ratio of the biotin-modified dNTP in the similar total dNTP is 10-100%;

preferably, the mixing molar ratio of the biotin-modified dNTPs in the similar total dNTPs is 20-50%;

and/or, the biotin-modified dNTP is a combination of two or all three of dATP, dCTP and dGTP;

preferably, the biotin-modified dNTP is a combination of dATP and dCTP.

7. The method of claim 2,

the length of the template is not uniform;

the primer is a single-ended primer and the 3' end of the primer is modified;

the single-stranded linear amplification is a multiple-round amplification.

8. The method of claim 2,

the amino acid sequence of the DNA polymerase is one or more of SEQ ID NO 1-SEQ ID NO 9; or the like, or, alternatively,

the homology of the amino acid sequence of the DNA polymerase and the N end of any one of SEQ ID NO. 1-SEQ ID NO. 9 with 360 amino acids is more than or equal to 90%.

9. The method of claim 1, further comprising the step of purifying the amplification product based on affinity purification of the tagged molecule, i.e., streptavidin magnetic bead purification.

10. Use of a method according to any one of claims 1 to 9 for amplifying a target nucleic acid.

Technical Field

The present invention relates to the field of biotechnology, and more particularly, to an optimized method and system for amplifying and purifying a target nucleic acid.

Background

For the targeted amplification technology, it is very critical that the target product with sufficient yield and purity is obtained after amplification and purification. The purification method of the target product after nucleic acid amplification in the prior art mainly comprises a solid phase carrier adsorption method, such as an adsorption column method or a solid phase reversible magnetic bead method, and utilizes the strong affinity and adsorption force of the carrier to nucleic acid; a molecular sieve method of screening target molecules by difference in molecular weight; an electrophoresis method in which nucleic acids having a target molecular weight are purified and recovered by electrophoretic analysis; and (3) affinity purification, namely labeling the probe or the primer by using a label, and purifying the target DNA molecule by utilizing the affinity of the label by using a solid phase carrier.

In view of throughput and cost, affinity purification based on the affinity of a label to a solid phase carrier is commonly used in the second generation sequencing library construction, such as streptavidin-coated magnetic beads to purify amplification products amplified by biotin-labeled probes or primers. Compared with the other three purification methods, the method has the advantages of high recovery rate of short fragments or single-stranded DNA, high conversion rate of the original DNA template, less non-nucleic acid products in the purified product and suitability for the original template with indefinite length, but has the defect that a large amount of free probes or primers with labeled molecules, which are not combined with the template, in the purified product are remained.

In order to solve the problem of a large amount of labeled free probes or primers remaining in the purified product, another purification method adopted in the prior art is to label a labeled molecule on dntps as a substrate for an amplification reaction instead of the probes or primers, incorporate the labeled dntps into the amplified product by an enzymatic reaction of DNA polymerase, and obtain the target purified product by affinity purification. In general, for each dNTP analog tested, there is an inverse relationship between the amplification product yield and the incorporation rate of the modified dNTP analog, and it is indispensable to find a suitably modified dNTP analog as a reaction substrate. However, the number of the natural dNTPs which can be modified by biotin is at least four, more than dozens of different sites of the four dNTPs are subjected to biotin modification with different carbon chain lengths, the substrate performances of different modified dNTP analogs are greatly different, and the product incorporation rate is still acceptable but the amplification rate is very low. Considering the problem of DNA polymerase, through the development of molecular biology till now, the subdivided types of the natural and directionally-modified DNA polymerase are more than enough, the structure and the performance of the DNA polymerase are different, different types of DNA polymerases are matched with different types of modifiers, modification sites and dNTP analogues with a mixing ratio, the arrangement and the combination are almost infinite, and the product yield and the purification performance are different from each other. The prior art only mentions that the DNA polymerase losing 3'-5' exonuclease activity can incorporate biotin-modified dNTP (Incorporation of reporter molecule-labeled nucleotides by DNA polymers. II. high-specificity labeling of native DNA, T Tasara, B anger, etc., Nucleic Acids Res.2003May 15; 31(10): 2636-2646.) in DNA amplification, but the DNA polymerase with inactivated 3'-5' exonuclease activity does not have high fidelity and cannot meet the requirement of targeted amplification technology for accurate enrichment of target Nucleic acid sequences. The B-type DNA polymerase can ensure the accuracy of amplified sequences, and the 3'-5' exonuclease activity of the B-type DNA polymerase ensures that the B-type DNA polymerase has higher base fidelity than other DNA polymerases. The prior art does not give any technical suggestion on how to amplify DNA of indefinite length by primer extension method while finding a suitable substrate while retaining the 3'-5' exonuclease activity of DNA polymerase, especially for the application scenario of the single-stranded linear amplification technique. The single-stranded linear amplification is a target nucleic acid amplification technology different from the traditional PCR, and the template nucleic acid is repeatedly combined and extended and amplified by adopting a single-ended primer, so that an amplification product is generated by linear amplification instead of exponential amplification, and the characteristic can avoid the accumulation of base errors introduced by the PCR and further ensure the accuracy of the amplification product.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention provides an optimized method for amplifying a target nucleic acid, comprising the steps of:

in a reaction system, target nucleic acid molecules are taken as templates, primers are added, a dNTP-DNA polymerase complex system with high doping rate is obtained, and amplification products of the target nucleic acids are obtained through amplification reaction;

wherein the high-incorporation-rate dNTP-DNA polymerase complex system comprises dNTP and DNA polymerase;

the dNTP is at least partially modified by a marker molecule, and a structure for preventing the modification group molecule from rotating cannot exist in the dNTP modified by the marker molecule;

the DNA polymerase is a high fidelity polymerase with 3'-5' exonuclease activity.

In another aspect, the present invention provides an optimized system for amplifying a target nucleic acid, comprising a labelled molecule modified dNTP analogue and a DNA high fidelity polymerase suitable for use in the optimized amplification method provided in the first aspect of the invention.

The present applicant has completed the present invention based on extensive efforts and expenses, and extensive trial and error studies to try a combination of an infinite number of enzymes and dNTP analogues, and to provide an optimized method for amplifying a target nucleic acid, which is simple in operation and excellent in performance, particularly, has an extremely superior product yield and incorporation efficiency for a single-stranded DNA product amplified by a single-ended primer having an indefinite template length.

In one aspect, the present invention provides an optimized method for amplifying a target nucleic acid, comprising the steps of:

in a reaction system, a target nucleic acid molecule is taken as a template, a primer and a dNTP-DNA polymerase complex system with high doping rate are added, and an amplification product of the target nucleic acid is obtained through an amplification reaction.

Wherein the high incorporation rate dNTP-DNA polymerase complex system comprises dntps and a DNA polymerase; wherein the dNTP is a dNTP at least partially modified by a labeling molecule, the DNA polymerase is a high-fidelity polymerase with 3'-5' exonuclease activity, and the amplification performed by the complex system is single-strand linear amplification. The high-doping-rate dNTP-DNA polymerase complex system can efficiently dope modified dNTP to obtain a high-purity target nucleic acid amplification product, solves the defect that the modified dNTP cannot be doped by 3'-5' exonuclease high-fidelity DNA polymerase, and effectively generates the high-purity high-fidelity target nucleic acid amplification product. With the modification optimization of the high-fidelity DNA polymerase, modified dNTP species with good doping effect are improved.

Wherein the DNA polymerase-C-terminus comprises a double-stranded DNA binding domain, Sso7 d; the C end can be a Pfu/Deep Vent chimera; may contain the V93Q mutation.

Wherein, when the DNA polymerase contains site-directed mutation of the N-terminal domain, the modification group in the dNTP modified by the marker molecule and the first 3C atoms connected with the dNTP base group contain or do not contain a double-bond or triple-bond structure for preventing the modification group molecule from rotating; when the DNA polymerase does not contain site-directed mutation of the N-terminal domain, the modification group in the dNTP modified by the marker molecule and the first 3C atoms connected with the dNTP base group cannot have a structure such as a double bond or a triple bond, and the like for preventing the molecule from rotating.

Further, when the DNA polymerase contains the V93Q mutation, the modifying group in the dNTP modified with the labeling molecule contains or does not contain a double or triple bond structure that prevents the modifying group molecule from rotating within the first 3C atoms to which the dNTP base group is attached; when the DNA polymerase does not contain the V93Q mutation, the modification group in the dNTP modified by the marker molecule and the first 3C atoms connected with the dNTP base group can not have a structure such as a double bond or a triple bond, etc. for preventing the molecule from rotating; the modification should not be near the ribose group at the point of attachment to the dNTP, i.e., the attachment site should not be C5 for 7-Deaza and dCTP of dATP/dGTP.

In the present invention, the high incorporation efficiency is defined as: the proportion of the dNTP modified by the marker molecule in the amplified product in the total dNTP exceeds 20 percent, and the purpose of purifying the amplified product can be effectively achieved.

Drawings

FIG. 1 is a structural diagram of Pfu enzyme, and the main functional structures in the present invention are an exouclase region (having a 3'-5' exonuclease function) common to class B DNA polymerases and a V93Q point mutation in the N-terminus.

FIG. 2 is a schematic diagram showing the structural composition of various DNA polymerases; wherein the content of the first and second substances,

1(SEQ1) Pfu as described in example 1 of the invention;

SEQ ID NO 2(SEQ2) de ep Vent as described in example 1 of the present invention;

3(SEQ3) which is KOD described in example 1 of the present invention;

SEQ ID NO 4(SEQ4) Pfu + Sso7d according to the invention (Add-on-tail 1, APO-1 in example 1);

SEQ ID NO 5(SEQ5) KOD + Sso7d according to the invention (plus tailpiece 2, i.e. APO-2 in example 1);

6(SEQ6) namely the Pfu/Deep Vent chimera of the invention + Sso7d (chimera 1, i.e. APO-3 in example 1);

SEQ ID NO:7(SEQ7), namely exchanger 1 according to the invention (i.e. APO-4 in example 1);

SEQ ID NO 8(SEQ8), namely heat exchanger 2 according to the invention (i.e. APO-5 in example 1);

SEQ ID NO 9(SEQ9), i.e.the exchanger 1+ V93Q point mutation according to the invention (i.e.APO-6 in example 1);

FIG. 3 is a sequence alignment display of various DNA polymerases; including the most important sequence of the invention, which has 3'-5' exonuclease activity and has the structure of about 300aa at the C end of the Pfu/Deep Vent chimera (i.e., the first 300aa of SEQ ID NOS: 7 and 9).

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.