CN106636071A - Method for synthesizing nucleic acid under constant-temperature condition - Google Patents
Method for synthesizing nucleic acid under constant-temperature condition Download PDFInfo
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- CN106636071A CN106636071A CN201710031322.6A CN201710031322A CN106636071A CN 106636071 A CN106636071 A CN 106636071A CN 201710031322 A CN201710031322 A CN 201710031322A CN 106636071 A CN106636071 A CN 106636071A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
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- Molecular Biology (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of gene engineering and in particular relates to a method for synthesizing nucleic acid under a constant-temperature condition. The method comprises the following steps: 1) providing a nucleic acid, wherein the 3' end of the nucleic acid has an F1r region for annealing with an F1c region on the same chain, and simultaneously annealing the F1r region and the F1c region to form a spiral ring; 2) synthesizing an own complementary chain by taking the nucleic acid of the step 1) as a template; 3) carrying out complementary chain synthesis through a polymerase catalytic chain replacement type complementary chain synthesis reaction, so as to replace the complementary chain synthesized in the step 2). According to the method provided by the invention, a nucleotide sequence provided at a 5'-side of a primer of oligonucleotide is basically the same as a region which is synthesized by taking the primer as a synthesis starting point. The nucleic acid synthesis based on a constant-temperature reaction is realized on the basis of the composition of a pure reagent.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a method for synthesizing nucleic acid under a constant temperature condition, in particular to a method for synthesizing nucleic acid which is composed of specific nucleotide sequences and can form a special structure, and a useful method for amplifying nucleic acid based on the specific nucleotide sequences.
Background
The most fundamental difference of organisms carrying genetic information of organisms, the analysis method based on nucleotide sequence complementarity can directly analyze the genetic characteristics carried by genes. This analysis is a very powerful method for identifying genetic diseases, canceration, microorganisms, etc. Since the target gene is not easily detected when the amount of the target gene in the sample is very small, the target gene must be amplified or its detection signal must be amplified. As a method for amplifying a target gene, the PCR method is considered to be the most classical method (Saiki, Gelfandet al 1988), and is also the most common technique for in vitro amplification of nucleic acid sequences. The exponential amplification result of the method ensures that the method has high sensitivity, establishes the position of the method in the field of molecular biological method detection, and develops the existing series of mature products after decades of development. In addition, the amplification product can be recycled and thus is widely used as an important tool for supporting genetic engineering techniques such as gene cloning and structure determination. However, the PCR method has obvious problems as follows: in actual operation, a special program temperature control system is required; the exponential rise of the amplification reaction makes it difficult to quantify; the sample and the reaction solution are susceptible to external contamination, and the problem of false positive is prominent.
The current human genome project has been completed, and Single Nucleotide Polymorphisms (SNPs) show the characteristics of large quantity and wide distribution, so that the analysis thereof is gradually emphasized. By designing primers so that their nucleotide sequences contain SNPs, detection of SNPs by means of PCR amplification is possible, i.e., by the presence or absence of a nucleotide sequence complementary to the primer can be inferred by the determination of the presence or absence of the reaction product. However, once the complementary strand is accidentally synthesized by mistake in PCR, the product is run as a template in the subsequent reaction, which may cause erroneous results. In practice, it is difficult to strictly control PCR if only one base at the end of the primer is different, so that it is necessary to improve the specificity so that PCR can be more preferably used for detection of SNPs.
On the other hand, compared with the synthesis of nucleic acid by a complicated programmed temperature-controlled process, scientists have developed a technique for synthesizing nucleic acid under constant temperature conditions (Zhao, Chen et al.2015), which mainly includes the following: nucleic acid sequence dependent amplification (NASBA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA), loop mediated isothermal amplification (LAMP), Helicase Dependent Amplification (HDA), Recombinant Polymerase Amplification (RPA).
NASBA, also known as TMA (transcription-mediated amplification method), does not require complex temperature control. The method is synthesized by adding a probe linked to a T7 promoter to a target RNA as a template by a DNA polymerase, allowing a second probe to enter into double strands to produce a product, and then transcribing the double-stranded DNA produced by the DNA polymerase to amplify the double-stranded DNA to produce a large amount of RNA product. NASBA requires a heat denaturation step until double-stranded DNA is formed, but the subsequent transcription reaction is performed by T7RNA polymerase under isothermal conditions. It is necessary to use a combination of various enzymes such as reverse transcriptase, RNase H, DNA polymerase and T7RNA polymerase, however, the combination of various enzymes is disadvantageous in terms of cost. Meanwhile, due to the complicated setting of reaction conditions of various enzymes, the method is difficult to popularize as a general analysis method.
RCA (Rolling Circle Amplification) is intended to mimic the process of Rolling Circle replication of circular DNA in microorganisms, and for circular single-stranded DNA templates, Amplification of circular nucleic acids can only be achieved in the in situ method using primers that bind to the template. To make this method universally applicable to amplification of linear DNA, single-stranded DNA complementary to a padlock probe (padlock probe) or a loop probe was shown to be continuously synthesized in the presence of a target nucleotide, with a series of nucleotide sequences complementary to the padlock probe (padlock probe) or the loop probe (Lizardi, Huang et al 1998). This method also has a problem that a plurality of enzymes are required. Moreover, the initiation of complementary strand synthesis depends on the reaction of joining two adjacent regions, and the specificity thereof is substantially the same as that in LCR.
The Strand Displacement Amplification (SDA) method is also known as a method for amplifying a template DNA having a sequence complementary to a target sequence (Zhang, Cui et al 1992). The SDA method employs a specific DNA polymerase to synthesize a complementary strand from a primer complementary to the 3 '-side of a target nucleotide sequence to replace the sequence on the 5' -side of the double strand. This technique is called SDA method because the newly synthesized complementary strand replaces the double strand on the 5' -side. The restriction enzyme recognition sequence as a primer inserted into the annealing sequence in the SDA method can remove the temperature change step necessary for the PCR method. That is, the 3' -OH group is supplied as the origin of complementary strand synthesis by the restriction enzyme-generated nick, and the complementary strand synthesized first is released as a single strand by strand displacement synthesis and then used again as a template for the next complementary strand synthesis. However, SDA amplification products differ in structure from the native nucleic acids and have limitations for use of restriction enzymes to break or apply the amplification products to gene clones. This aspect is also the main reason for the higher cost. In addition, when the SDA method is applied to an unknown sequence, a nucleotide sequence identical to the recognition sequence of the restriction enzyme for introducing a nick may be present in the region to be synthesized, and thus synthesis of a completely complementary strand may be prevented.
Helicase-dependent Isothermal DNA amplification (HAD) is a novel Isothermal nucleic acid amplification technique invented by researchers of the American NEB company in 2004 (Vincent, Xu et al 2004). The technology simulates the natural process of DNA replication in a natural organism, unwinding a DNA double strand by helicase under the condition of constant temperature, simultaneously, stably unwinding a single strand by single-stranded DNA-binding protein (SSB), providing a binding template for a primer, and then catalytically synthesizing a complementary strand by DNA polymerase. The newly synthesized double strand is decomposed into single strand under the action of helicase, and the single strand is used as a template for the next round of synthesis to enter the cyclic amplification reaction, and finally the exponential growth of the target sequence is realized.
At the heart of LAMP technology (Notomi, Okayama et al 2000), four specific primers are designed for six regions on the target gene, relying on a highly active strand displacement DNA polymerase, so that strand displacement DNA synthesis is constantly self-cycling. The technology can realize 10 in 15-60 minutes9-1010Amplification of the foldThe reaction can produce a large amount of amplification products, i.e. white magnesium pyrophosphate precipitate, and can judge whether the target gene exists by observing the existence of the white precipitate with naked eyes, and Japan Rongyan company also develops a turbidimeter in a targeted manner to realize the real-time monitoring of the amplification reaction. The LAMP method has the advantages of high specificity and high sensitivity, is very simple to operate, has low requirements on instruments in the application stage, can realize reaction by using a simple constant temperature device, is very simple in result detection, can be used for directly observing white precipitates or green fluorescence by naked eyes, and is a method suitable for rapid detection on site and in basic level. One of the limitations is that the method relies on the properties of 4 primers for its high specificity and sensitivity, and the acquisition of the optimal primers usually requires sequence comparison, on-line primer design, primer screening and specificity test, which is a very tedious process.
Recombinase Polymerase Amplification (RPA), whose main point is: the recombinase, in combination with the primer, forms a protein-DNA complex that is able to search for homologous sequences in double-stranded DNA. Once the primers locate the homologous sequences, the strand-displacing DNA polymerase then mediates the formation of strand-exchange reactions and initiates DNA synthesis, which exponentially amplifies the target region on the template. The replaced DNA strand binds to a single strand binding protein (SSB) to prevent further replacement. In this system, a single synthesis event is initiated by two opposing primers. The entire process is carried out very quickly and detectable levels of amplification product are typically obtained within ten minutes. However, the need to select primers that bind to the recombinase and have good specificity in the whole process, and the need to use three enzymes greatly increases the cost.
Liuwei et al (Liu, Dong et al 2015) of the military medical academy of sciences of the liberation military, proposed a novel nucleic acid isothermal amplification technique called polymerase helix reaction (PSR), which uses the same enzyme as LAMP reaction, and by introducing a specific sequence into the 5 ' end of a designed PCR primer for amplification to form a structure in which the 5 ' and 3' ends can self-assemble into a closed helix to provide 3' -OH as a starting point for synthesis and use the 3' -OH as an amplification starting structure, then a large number of DNA single strands of the same sequence are synthesized, and finally a long-chain helix structure product is formed. However, the method introduces a foreign gene into the primer sequence, which greatly increases the possibility of mismatch and leads to erroneous results. In addition, the loop overlapping part in the closed loop structure in the initial structure is only about 20 base pairs, which weakens intermolecular hydrogen bonds to further make the helical loop unstable, and at the same time, if the length of the exogenous gene is too long, the helical loop forming process is affected.
Disclosure of Invention
The invention aims to provide a method for synthesizing nucleic acid, which is inspired by the common double helix structure of DNA and LAMP and PSR methods, redesigns and plans a primer working mode on the basis of PCR primers, amplifies an initial structure by a helix loop, designs the forming process of the structural loop, and has the new characteristics of more base pairs of an overlapping ring forming part, no introduction of exogenous gene fragments and reusability of amplified products. More particularly, to provide a novel, low-cost method for efficiently synthesizing nucleic acids by means of sequences. That is, the object of the present invention is to provide a method for synthesizing and amplifying nucleic acids by a single enzyme under isothermal conditions. Another object of the present invention is to provide a method for synthesizing nucleic acid, which can synthesize nucleic acid rapidly with high specificity, which is difficult to achieve by modifying the existing principles of nucleic acid synthesis reaction, and a method for amplifying nucleic acid using the synthesis method.
The present invention utilizes polymerase to catalyze strand displacement-type complementary strand synthesis without complicated temperature control, and is useful for nucleic acid synthesis. The DNA polymerase is an enzyme used in methods such as SDA, RCA, LAMP and PSR.
The present inventors improved the supply of 3 '-OH in the known method, and as a result, found that by using an oligonucleotide having a specific structure, the 3' -OH structure can be provided without any additional enzymatic reaction, thereby leading to the present invention. Namely, the present invention relates to a method for synthesizing a nucleic acid, a method for amplifying a nucleic acid by using the nucleic acid synthesis method, and a kit for applying the method.
The invention has the technical scheme as follows:
the method for synthesizing nucleic acid under the constant temperature condition comprises the following steps:
1) providing a nucleic acid having at its 3' end a F1r region that anneals to a F1c region on the same strand and a helical loop formed by the simultaneous annealing of the F1r region to a Flc region;
2) synthesizing a complementary strand of the nucleic acid of the step 1) by using the nucleic acid as a template, wherein the R1rc region of the complementary strand is annealed with the R1 region, and the 3' end of the F1R region annealed by the Flc region forms a spiral loop and is used as a synthesis starting point;
3) complementary strand synthesis is performed by polymerase-catalyzed strand displacement-type complementary strand synthesis reaction to displace the complementary strand synthesized in step 2), wherein the polynucleotide comprises at its 3' end a sequence complementary to any region of the complementary strand synthesized in step 2).
The method for synthesizing nucleic acid according to the present invention, wherein the method for preparing nucleic acid of step 1) comprises the steps of:
i) an annealing step of annealing a first oligonucleotide I to a F1c region of a template, wherein the 3' end of the template comprises a F1c region and a F2c region located 3' to the F1c region and the 5 ' end of the template comprises a R1 region and a R2 region located 5 ' to the R1 region, wherein said first oligonucleotide I, comprising a R1R region and a Fl region, said Rlr region is linked to the 5 ' to the F1 region, wherein,
region F1: a region having a nucleotide sequence complementary to the region of the template F1c,
R1R region: a region opposite to the region of template R1;
ii) a step of synthesizing a first nucleic acid having a nucleotide sequence complementary to the template, the step synthesizing the first nucleic acid with the F2 region of the first oligonucleotide I as a starting point of synthesis, the 3' -end of the first nucleic acid having an F1 region annealable to a portion F1c region on the same strand, and a helical loop formable by simultaneous annealing of the F1 region and a Flc region;
iii) displacing the first nucleic acid synthesized in step ii) with a polymerase-catalyzed strand displacement reaction in which the first outer primer I annealing to the F2c region 3' to F1c in the template serves as the start of synthesis, and
iv) an annealing step of annealing a second oligonucleotide II to the R1c region of the first nucleic acid obtained in step iii), wherein the second oligonucleotide II comprises a R1 region and a F1R region, and a Flr region is linked to the 5' side of the R1 region, the second oligonucleotide II being the reverse sequence of the first oligonucleotide I; wherein,
region R1: a region having a nucleotide sequence complementary to the R1c region of the first nucleic acid,
flr area: a region that is inverted with respect to the F1 region of the first nucleic acid;
v) synthesizing a second nucleic acid with the second oligonucleotide II as the synthesis starting point, and replacing the second nucleic acid by a polymerase-catalyzed strand displacement reaction to obtain the nucleic acid of step 1); wherein the second outer primer II annealing to the R2c region 3' to R1c in the first nucleic acid serves as the origin of synthesis when the second nucleic acid is replaced.
Further, the template in step i) is RNA and the first nucleic acid in step ii) is synthesized by an enzyme having reverse transcriptase activity. The present invention is applicable to various RNAs such as various viral RNAs and the like. For example, for RNA detection of MERS-CoV virus, such as: orf1a, orf1b segment of the RNA, and the like.
In the process of forming the initial helix structure, the oligonucleotide used in the present invention may have no sequence connecting the F1 region with the R1R region, or connecting the R1 region with the F1R region, but if a linker (L) is introduced, the sequence must be an inverted sequence, i.e., the two oligonucleotides are 5 '-R1R-L-F1-3', 5 '-F1R-Lr-R1-3', L and Lr must be reverse complementary sequences (R1R is the inverted sequence of R1 and F1R is the inverted sequence of F1), and the linker is generally a restriction endonuclease site sequence.
The method for synthesizing a nucleic acid according to the present invention, wherein the synthesized nucleic acid is a nucleic acid having a nucleotide sequence complementary end to end on one strand thereof.
The method for synthesizing nucleic acid according to the present invention, wherein the constant temperature means that the synthesis is performed at a constant temperature of 60-65 ℃ throughout the reaction process.
The method for synthesizing nucleic acid according to the present invention, wherein the melting temperature between each oligonucleotide and its complementary region in the template has the following relationship under the same stringent conditions: (outer primer or template 3' side region) of ≦ (F2c or F2, and, R2c or R2) ≦ (F1c or F1, and, Rlc or Rl).
According to the method for synthesizing a nucleic acid of the present invention, preferably, the obtained second nucleic acid can be accelerated in nucleic acid amplification by introducing an acceleration probe Xin, wherein Xin is an intermediate region from the F1 region to the R1 region.
The invention can repeat the steps 1) -3) by using the complementary strand replaced in the step 3) as a template to synthesize the long-chain nucleic acid.
The polymerase used in the polymerase catalytic strand displacement reaction is one or more of Bst DNA polymerase, Bca (Exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (Exo-) DNA polymerase (Vent DNA polymerase lacking exonuclease activity), Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase (Deep Vent DNA polymerase lacking exonuclease activity), phi 29phage DNA polymerase, MS-2phageDNA polymerase and the like. Among them, Bst DNA polymerase or Bca (exo-) DNA polymerase is preferably used.
The method for synthesizing nucleic acid according to the present invention, wherein a melting temperature regulator may be added to the polymerase-catalyzed strand displacement reaction. Wherein the melting temperature regulator is preferably betaine, and further preferably, the concentration of betaine in the reaction solution is allowed to be 0.2 to 3.0M.
The kit for synthesizing nucleic acid under isothermal conditions according to the present invention is characterized by comprising:
an oligonucleotide I comprising a F1 region and a R1R region, said R1R region being linked to the 5' side of the F1 region, wherein,
region F1: a region having a nucleotide sequence complementary to the F1c region of the template, and
R1R region: a region opposite to the region R1 of the template;
an oligonucleotide II comprising a region R1 and a region F1R, said region F1R being linked to the 5' side of the region R1, wherein,
region R1: a region having a nucleotide sequence complementary to the R1c region of the template, and
f1 r: a region opposite to the F1 region of the template;
a first primer I having a nucleotide sequence complementary to the F2c region 3' to the F1c region in the nucleic acid as a template;
a second primer II having a nucleotide sequence complementary to the R2c region 3' to the R1c region in the nucleic acid as a template;
a DNA polymerase catalyzing a strand displacement-type complementary strand synthesis reaction, and,
a nucleotide that serves as a substrate for the DNA polymerase.
The kit according to the present invention, wherein the kit further comprises a detection reagent for detecting a product of the nucleic acid synthesis reaction.
The kit according to the present invention, wherein the kit further comprises an acceleration probe Xin, wherein Xin is a nucleotide fragment located in the middle segment from the F1 region to the R1 region of the template, specifically, the Fin and Rin regions located between F1 and R1.
According to the kit, the DNA polymerase is one or more of Bst DNA polymerase, Bca (Exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (Exo-) DNA polymerase, Deep Vent (Exo-) DNA polymerase, phi 29phage DNA polymerase, MS-2phage DNA polymerase and the like. Among them, Bst DNA polymerase or Bca (exo-) DNA polymerase is preferably used.
The invention provides application of the kit in synthesizing nucleic acid or detecting a target nucleotide sequence in a sample.
Based on the method for synthesizing a nucleic acid of the present invention, there is provided a method for detecting a target nucleotide sequence in a sample, comprising amplifying by the method for synthesizing a nucleic acid of the present invention using a target nucleotide as a template, and observing whether or not an amplification product is produced.
A probe comprising a nucleotide sequence complementary to the formed helical loop is added to the amplification product, and hybridization between the two is observed. The probe may also be labeled on a particle and the aggregation reaction by hybridization observed. The amplification method may be carried out in the presence of a nucleic acid detection reagent, and whether or not an amplification product is produced is observed based on a change in a signal of the detection reagent.
Based on the method for synthesizing nucleic acid of the present invention, there can also be provided a method for detecting a mutation in a target nucleotide sequence in a sample, comprising amplifying by the method for synthesizing nucleic acid of the present invention using the target nucleotide as a template. Wherein a mutation to be amplified in the nucleotide sequence inhibits synthesis of any of complementary strands constituting the amplification method, and the mutation is detected.
A single-stranded nucleic acid having nucleotide sequences complementary at the head and the tail is the object of the synthesis of the present invention, and refers to a nucleic acid having a target nucleic acid sequence linked in a single strand with mutually complementary nucleotide sequences in the head and side by side. In addition, a nucleotide sequence for forming a helix between complementary strands should be included in the present invention. This sequence is referred to herein as a helical loop sequence. The nucleic acids synthesized in the present invention consist essentially of mutually complementary strands connected by a helical loop-forming sequence. In general, a strand that cannot be separated into two or more molecules upon base pairing separation is referred to as a single strand, regardless of whether a portion is involved in base pairing. Complementary nucleotide sequences in the same strand can form base pairing, and the present invention can obtain an intramolecular base-paired product comprising a region constituting a significant double strand and a loop not involved in base pairing by allowing nucleic acids having nucleotide sequences joined end to end in a single strand to base pair within the same strand.
That is, the nucleic acid of the present invention having a complementary nucleotide sequence joined end to end in a single strand can be defined as a single-stranded nucleic acid comprising a complementary nucleotide sequence capable of annealing in the same strand, and the annealing product thereof, in the bent portion, constitutes a loop not involved in base pairing. Nucleotides having a complementary nucleotide sequence can anneal into loops not involved in base pairing. The loop forming sequence may be any nucleotide sequence. The loop-forming sequences are capable of base pairing to initiate synthesis of the complementary strand for displacement. And is preferably provided with a sequence different from the nucleotide sequence located in the other region to obtain specific annealing.
The nucleotide sequences that are substantially identical in the present invention are defined as follows: when a complementary strand synthesized with a certain sequence as a template anneals to a target nucleotide sequence to supply the origin of synthesizing the complementary strand, the certain sequence is substantially identical to the target nucleotide sequence. For example, a sequence substantially identical to F1 includes not only the sequence identical to F1 entirely but also a nucleotide sequence capable of serving as a template that gives a nucleotide sequence to which F1 anneals and can serve as a starting point for synthesizing a complementary strand. The term "annealing" according to the present invention refers to nucleic acids that form complementary structures by base pairing according to Watson-Crick's law. Therefore, even if a nucleic acid strand constituting base pairing is single-stranded, annealing occurs if the intramolecular complementary nucleotide sequence base pairs. Since the double-stranded structure is formed by base-pairing nucleic acids, the meaning expressed by annealing and hybridization according to the present invention is a coincidence.
The number of nucleotide sequence pairs constituting a nucleic acid of the present invention is at least 1. In the model contemplated by the present invention, the nucleotide sequence number of pairs may be an integer multiple of 1. In this case, there is no upper limit in the theoretical logarithm of the complementary nucleotide sequence of the constituent nucleotides of the present invention, and in the case of the synthetic product nucleic acid of the present invention composed of a plurality of sets of complementary nucleotide sequences, the nucleic acid is composed of nucleotide sequences that are identical in repetition.
The single-stranded nucleotides synthesized in the present invention having a head-to-tail complementary loop sequence may not have the same structure as a naturally occurring nucleic acid, and it is generally known that a nucleic acid derivative can be synthesized if a nucleotide derivative is used as a substrate when synthesizing a nucleic acid by the action of a nucleic acid polymerase. The nucleotide derivatives used include radioisotope-labeled nucleotides or nucleotide derivatives labeled with a binding ligand such as biotin or digoxigenin. These nucleotide derivatives are useful for labeling product nucleic acids. Alternatively, if the substrate is a fluorescent nucleotide, the product nucleic acid may be a fluorescent derivative. And the product may be DNA or RNA. The product produced is determined by combining the structure of the primer for realizing the nucleic acid polymerization reaction, the type of the substrate for the polymerization reaction and the reagent for the polymerization reaction.
The synthesis of a nucleic acid having the above structure can be initiated by using a DNA polymerase having a strand displacement activity and having a 3' -end in which the F1r region anneals to a part of the Flc region on the same strand to form a synthetic complementary strand. There are many reports on complementary strand synthesis reactions in which hairpin loops are formed using the hairpin loop sequence itself as a template and helical loops are formed using the helical loop sequence itself as a template, and the present invention provides a region of the helical loop whose head and tail portions can base pair, and has a novel feature of utilizing this region in synthesizing a complementary strand. By using this region as the origin of synthesis, the complementary strand previously synthesized with the helical loop sequence itself as a template is replaced.
The present invention uses the term "nucleic acid", which typically includes both DNA and RNA. However, nucleic acids or modified nucleotides from natural DNA or RNA whose nucleotides are replaced by artificial derivatives, which function as templates for the synthesis of complementary strands, are also included in the scope of the nucleic acids of the invention. Typically, the nucleic acids of the invention are contained in biological samples, including tissues, cells, cultures and secretions of animals, plants or microorganisms, as well as extracts thereof. The biological sample of the invention comprises intracellular parasite genomic DNA or RNA, such as a virus or mycoplasma. The nucleic acids of the invention are generally derived from the nucleic acids contained in said biological sample. For example, cDNA synthesis from mRNA, nucleic acids amplified based on nucleic acids derived from biological samples, are typical examples of nucleic acids of the present invention.
The nucleic acid of the present invention is characterized in that the F1R region is provided at the 3' -end, and can anneal to a part of Flc on the same strand, and a loop including a base-pairable R1 region can be formed by annealing the F1R region to Flc on the same strand, and the nucleic acid can be obtained in various methods.
The terms "identical" and "complementary" used to make up the nucleotide sequence features based on the oligonucleotides of the invention do not imply absolute identity and absolute complementarity. That is, a sequence identical to a certain sequence includes a sequence complementary to a nucleotide sequence to which the certain sequence anneals. On the other hand, the complementary sequence is a sequence that can anneal under stringent conditions and is provided as the 3' -end of the origin of complementary strand synthesis.
In the present invention, an oligonucleotide is a nucleotide that satisfies two requirements, i.e., must be capable of forming complementary base pairing and supply an-OH group at the 3' -end as the origin of complementary strand synthesis. Therefore, the main chain thereof is not necessarily limited to phosphodiester bond-type linkage. For example, it may consist of a backbone of phosphorothioate derivatives which are S substituted for O or be peptide nucleic acids based on peptide linkages. Bases are those bases that can be complementarily paired. Five bases, namely a, C, T, G and U, occur naturally, and bases may also be analogs such as bromodeoxyuridine. Preferably, the oligonucleotide of the present invention can be used not only as an origin of synthesis but also as a template for complementary strand synthesis. The term polynucleotide of the present invention includes oligonucleotides. The term "polynucleotide" as used herein is not limited in its chain length, while the term "oligonucleotide" as used herein refers to a polymer of nucleotides having a relatively short chain length.
The oligonucleotide chain of the present invention has such a length that it can base-pair with a complementary chain and maintain a certain specificity under given conditions in various nucleic acid synthesis reactions described below. Specifically, it consists of 5 to 200 bases, more preferably 10 to 50 base pairs. The chain length recognizing the known polymerase is at least 5 bases. The polymerase catalyzes a sequence-dependent nucleic acid synthesis reaction. The chain length of the annealed portion should be longer than this length. In addition, a length of 10 bases or more is statistically expected to obtain the target nucleotide specificity. On the other hand, it is difficult to prepare a too long nucleotide sequence by chemical synthesis. Thus the above chain lengths are examples of desirable ranges. Exemplary chain lengths refer to those lengths that partially anneal to a complementary strand. As described below, the oligonucleotides of the invention can eventually anneal to at least two regions, respectively. Thus, chain lengths exemplified herein are to be understood as the chain length of each region that makes up the oligonucleotide.
Furthermore, the oligonucleotide of the present invention may be labeled with a known label. Labels include binding ligands such as digoxigenin and biotin, enzymes, fluorescers, luminophores, radioisotopes. The technique of replacing the bases constituting an oligonucleotide by fluorescent analogs is well known (W095/05391, Proc. Natl. Acad. Sci. USA, 91, 6644-.
Other oligonucleotides of the invention may also be bound to a solid phase. Alternatively, any part of the oligonucleotide may be labelled with a binding ligand, such as biotin, which is indirectly immobilised by the binding ligand, such as immobilised avidin. When the immobilized oligonucleotide is the starting point of synthesis, the nucleic acid of the product of the synthesis reaction is captured by a solid phase, which facilitates its isolation. The separated portions may be detected by nucleic acid specific indicators or hybridization to labeled probes. The nucleic acid product obtained by the present method is directed to a method wherein the target nucleic acid fragment is recovered by digestion of the product with a restriction enzyme.
The term "template" as used herein refers to a nucleic acid that serves as a template for the synthesis of a complementary strand. The complementary strand having a nucleotide sequence complementary to the template means a strand corresponding to the template. But the relationship is relative. I.e., the synthesized complementary strand can function as a template again. That is, the complementary strand may also serve as a template.
In the present invention, if the target is RNA, it can be constituted by adding only reverse transcriptase, i.e., RNA as a template, by annealing F1 to F1c in the template by reverse transcriptase it is possible to synthesize a complementary strand and to synthesize a complementary strand from the point of annealing to F2c outer primer F2 for synthesis and to replace the previously synthesized complementary strand at the same time, outer primer F2 being located on the 3' -side of F1 c. When the reverse transcriptase carries out a reaction of synthesizing a complementary strand using DNA as a template, all reactions of synthesizing a complementary strand by the reverse transcriptase include the synthesis of a complementary strand using R1 annealed to Rlc as a synthesis origin, which serves as a template in a strand displacement reaction; synthesis of a complementary strand with R2 annealed to R2c as a synthesis origin and simultaneous strand displacement reaction, R2 is located on the 3' -side of R2 c. When the reverse transcriptase cannot be expected to exhibit DNA/RNA strand displacement activity under the given reaction conditions, a DNA polymerase having the strand displacement activity as described above may be bound. The mode of obtaining the first single-stranded nucleic acid using RNA as a template as described above is a preferred mode of the present invention. On the other hand, if a DNA polymerase having both strand displacement activity and reverse transcriptase activity, such as Bca DNA polymerase, is used, the synthesis from not only the first single-stranded nucleic acid of RNA but also the subsequent reaction using DNA as a template can be similarly performed by the same enzyme.
The reaction is carried out in the presence of a buffer that allows the enzyme reaction to be at a suitable pH, salts necessary to anneal or maintain the enzymatic activity, mediators to protect the enzyme, and modulators necessary to control the melting temperature (Tm). As buffers, for example Tris-HCl is used which has a buffering action in the neutral or weakly alkaline range. Adjusting the pH according to the DNA polymerase used, for salt, KCl, NaCl, (NH4)2SO4And adding proper amount to maintain the activity of the enzyme and regulate the melting temperature (Tm) of nucleic acid, wherein bovine serum albumin or saccharide is used as a medium for protecting the enzyme. In addition, dimethyl sulfoxide (DMSO) or formamide are typically used as regulators of the melting temperature (Tm). The regulation of annealing of oligonucleotides under defined temperature conditions is achieved by using a regulator of melting temperature (Tm). Furthermore, betaine (N, N, N-trimethylglycine) or tetraalkylammonium salts (tetraalkyl) are improved by their isostabilization (isotabinization)The efficiency of strand displacement is also effective. The desired promoting effect of the present invention on nucleic acid amplification can be obtained by adding 0.2 to 3.0M betaine, preferably 0.5 to 1.5M betaine, to the reaction solution. Since these melting temperature regulators have the function of lowering the melting temperature, those suitable stringent and reactive conditions are empirically determined in terms of the concentration of the binding salt, the reaction temperature, etc.
An important feature of the present invention is that a series of reactions cannot proceed unless the positional relationship of many regions is maintained. Due to this feature, the non-specific synthesis reaction accompanying the non-specific synthesis of the complementary strand is effectively prevented. That is, even if a certain nonspecific reaction occurs, the possibility that the product will serve as a starting material in the subsequent amplification step of the synthesis is reduced, and, by regulating the progress of the reaction through many regions, it is possible to allow a detection system that can accurately identify the desired product in a similar nucleotide sequence to be composed arbitrarily.
The nucleic acid synthesized in the present invention is a single strand, and in terms of single strand, is composed of complementary nucleotide sequences, most of which are base-paired. By utilizing this feature, the synthesized product can be detected. By carrying out the method for synthesizing a nucleic acid of the present invention, an increase in the intensity of fluorescence is observed with an increase in the product in the presence of a fluorescent dye as a double-strand specific intercalator (e.g., ethidium bromide, SYBR Green I, Pico Green or Eva Green). By monitoring the fluorescence intensity, it is possible to track the progress of real-time (real-time) synthesis reactions in a closed system. It is also conceivable to apply this type of detection system in the PCR method, but there are many problems because it is impossible to distinguish between a product signal and a signal of primer-dimer, etc. However, when the present invention employs this system, the ability to increase non-specific base pairing is very low, and therefore, it is expected that high sensitivity and low interference may be simultaneously obtained, and a method for realizing a detection system using the transfer of fluorescence energy in the same system, similarly to the use of a double-strand-specific intercalator (double-strand-specific intercalator).
The method of synthesizing a nucleic acid of the present invention is supported by a complementary strand reaction of strand displacement type catalyzed by DNA polymerase. The reaction period also includes a reaction step in which a strand displacement-type polymerase is not required. However, in order to make up the reagent simple and from the economical viewpoint, it is advantageous to use a DNA polymerase, the following enzymes are known. Furthermore, various mutants of these enzymes, all having sequence-dependent activity and strand displacement activity for complementary strand synthesis, can be utilized within the scope of the present invention. Where mutants are meant to include those having only the structure that results in the desired catalytic activity of the enzyme or those modified for catalytic activity, stability or thermostability by, for example, mutation in an amino acid.
Bst DNA polymerase
Bca (exo-) DNA polymerase
DNA polymerase I Klenow fragment
Vent DNA polymerase
Vent (Exo-) DNA polymerase (Vent DNA polymerase lacking exonuclease activity)
Deep Vent DNA polymerase
Deep Vent (Exo-) DNA polymerase (Deep Vent DNA polymerase lacking exonuclease activity)
Phi 29 phase DNA polymerase
MS-2 phase DNA polymerase
Of these enzymes, Bst DNA polymerase or Bca (exo-) DNA polymerase is particularly desirable because of its certain degree of thermal stability and high catalytic activity. In a preferred embodiment, the reaction of the present invention can be carried out isothermally, but it is not always possible to maintain the stability of the enzyme using a desired temperature condition due to adjustment of melting temperature (Tm) and the like. Therefore, it is one of the conditions required for the thermal stabilization of the enzyme. Although isothermal reactions are feasible, thermal denaturation can provide the nucleic acid as the initial template, in this regard, the use of thermostable enzymes broadens the choice of assay protocols.
The various reagents necessary for synthesizing or amplifying a nucleic acid of the present invention may be prepackaged and provided in the form of a kit, and specifically, the kit provided by the present invention comprises various oligonucleotides necessary as primers for synthesizing complementary strand synthesis and outer primers for displacement reaction, dntps as substrates for complementary strand synthesis, DNA polymerase for effecting strand displacement-type complementary strand synthesis, buffers for providing appropriate conditions for the enzymatic reaction, and media necessary for detecting the products of the synthesis reaction. In particular, in a preferred mode of the invention, no reagents need to be added during the reaction and thus the reagents necessary to be supplied for the subsequent reaction after transfer into the reaction vessel, wherein the reaction can be initiated by the addition of the sample alone. A system for detecting a reaction product in a container by using a visible light signal or a fluorescent signal. The vessel does not have to be opened and closed after the reaction. This is very advantageous for preventing contamination.
The invention synthesizes single-stranded nucleic acid with nucleotide sequence of which the head sequence and the tail sequence can anneal into a ring. The nucleic acid has, for example, the following uses: the first feature is the advantage of using a specific structure having a complementary sequence in a molecule, which may facilitate detection, i.e., there are known systems for detecting nucleic acids in which the signal to change depends on base pairing with a complementary nucleotide sequence. For example, by combining the methods using a double-strand specific intercalator as a detection reagent as described above, a detection system that takes full advantage of the characteristics of the synthesized product of the present invention can be realized. If the product of the synthetic reaction of the invention is heat denatured once in the detection system and returned to the original temperature, intramolecular annealing occurs preferentially and thus allows rapid base pairing between complementary sequences. If non-specific products are present, they have no complementary sequence in the molecule so that they cannot return to the original double strand immediately after separation into 2 or more molecules by heat denaturation. Interference accompanying non-specific reactions is reduced by the heat denaturation step provided prior to detection. If the DNA polymerase used is not resistant to heat, the heat denaturation step has the meaning of reaction termination and thus it is advantageous to control the reaction temperature.
The second feature is that closed loops are often formed that can base pair end-to-end. The structure of the loop capable of base pairing is shown in FIG. 3. The loop, as seen in FIG. 3, is composed of the nucleotide sequences F1, R1, F1c, R1c and undergoes intramolecular annealing to form a closed loop.
According to a preferred mode of the present invention, a large number of loops capable of base pairing are provided in a single-stranded nucleic acid. This means that a large number of probes can hybridize to a molecular nucleic acid to allow for highly sensitive detection. It is thus possible to realize not only improved sensitivity but also a method for detecting nucleic acids based on a special reaction principle such as aggregation. For example, a probe immobilized on a fine particle such as polystyrene latex is added to the reaction product of the present invention, and aggregation of the latex particle is observed as hybridization of the product with the probe. The intensity of the aggregation can be observed with high sensitivity and quantitatively by optical measurement. Alternatively, the aggregation can also be observed by the naked eye, so that a reaction system without an optical measuring device can also be established.
Furthermore, the reaction products of the invention allow for some bindable labels, wherein each nucleic acid molecule can be detected chromatographically. In the field of immunoassays, analytical methods using chromatographic media with visible detection markers (immunochromatography) are in practical use. This method is based on the principle that an analyte is sandwiched between an antibody immobilized on a chromatography medium and a labeled antibody, and an unreacted labeled component is eluted. The reaction products of the present invention apply this principle to nucleic acid analysis. That is, a labeled probe for the loop portion is prepared and immobilized on a chromatographic medium to prepare a capture probe for capture to allow analysis in the chromatographic medium. Capture probes whose sequence is complementary to the loop portion are used, since the reaction product of the present invention has a large number of loops, and the product binds to a large number of labeled probes to give a visually recognizable signal.
The reaction products of the present invention are often capable of providing base-paired loop regions, allowing a wide variety of other detection systems. For example, a system for detecting the loop portion using the surface plasmon using an immobilized probe is available. Furthermore, if the probe of the loop portion is labeled with a double-strand specific intercalator, a more sensitive fluorescence analysis can be performed. Or positively utilize the ability of the present invention to synthesize nucleic acids on the 3 '-and 5' -sides to form a helical loop capable of base pairing. For example, one loop is designed to have a common nucleotide sequence between the normal and abnormal types, while the other loop is designed to make a difference therebetween. The presence of the gene in the common part is confirmed by the probe, and when the abnormal presence is confirmed in the other region, it is possible to constitute a characteristic analysis system. Since the reaction for synthesizing a nucleic acid of the present invention can be carried out isothermally, it is worth mentioning an advantage that real-time analysis can be carried out by a general fluorescence photometer. Until this time, the structure of the nucleic acid to be annealed in the same strand was known. However, the nucleic acid in the single-stranded sequence having a loop formed by head-to-tail annealing obtained by the present invention is novel and contains a large number of loops capable of base-pairing with other bases.
On the other hand, a large number of loops given by the reaction product of the present invention can be used as probes themselves, for example, in a DNA chip, probes are packed in a limited region in a high density, and the number of oligonucleotides that can be immobilized in a certain region is limited in this technique, so that a large number of probes that can be annealed can be immobilized in a high density by using the product of the present invention, that is, the reaction product of the present invention can be used as immobilized probes on a DNA chip, and the reaction product after amplification can be immobilized by any technique known in the art, or immobilized oligonucleotides can be used as oligonucleotides for the amplification reaction of the present invention, resulting in the formation of immobilized reaction products. Thus, by using the immobilized probe, a large amount of sample DNA is hybridized in a limited region, and as a result, a high signal value is expected.
Drawings
FIG. 1 is a schematic diagram of the steps of the present invention for synthesizing a first nucleic acid.
FIG. 2 is a schematic diagram of the steps for synthesizing a second nucleic acid according to the present invention.
FIG. 3 is a diagram illustrating a loop structure formed by a single-stranded nucleic acid of the present invention.
FIG. 4 is a graphical representation of the loop structure formed by a single-stranded nucleic acid of the invention accelerated by the addition of additional primers.
FIG. 5 is a diagram showing the positional relationship of each nucleotide sequence constituting an oligonucleotide in the MERS-orf1b target nucleotide sequence.
FIG. 6 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid of the present invention using MERS-orf1b as a template.
Lane 1: biyuntian O0107DNA Ladder
Lane 2: 1fmol MERS-orf1b dsDNA
FIG. 7 is a diagram showing the positional relationship of each nucleotide sequence constituting an oligonucleotide in the MERS-orf1b target nucleotide sequence.
FIG. 8 is a photograph showing the result of agarose gel electrophoresis of a restriction enzyme digestion product obtained in example 1 by the nucleic acid synthesis reaction of the present invention. Wherein,
lane 1: biyuntian O0107DNA Ladder;
lane 2: 1fmol MERS-orf1b dsDNA;
lane 3: HindIII digestion of the purified product.
FIG. 9 is a real-time fluorescence curve showing the increase of DNA containing the MERS-orf1b target nucleotide sequence by the primer.
FIG. 10 is a real-time fluorescence curve showing the increase of DNA containing the MERS-orf1b target nucleotide sequence by the primer.
FIG. 11 is a graph showing the fluorescence intensity of amplification systems with different DNA target concentrations as a function of reaction time by adding an accelerating primer under the action of a primer.
FIG. 12 is a schematic flow chart of a method for synthesizing a nucleic acid of the present invention.
FIG. 13 is a schematic diagram of a nucleic acid helix synthesized by the present invention.
Detailed Description
The experimental procedures used in the following examples are conventional unless otherwise specified, and may be specifically carried out by the methods specified in molecular cloning, a laboratory manual (third edition) j. sambrook, or according to kits and product instructions; materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 amplification of the fragment in MERS-orf1b
The nucleic acid of the present invention having a complementary strand ligated into a single strand in the form of a helical loop was attempted using MERS-orf1b (from GenBank: NM-001012270.1) as a template. Four primers, Mo1bHF, Mo1bHR, Mo1bF2 and Mo1bR2 were used in the experiment. Mo1bF2 and Mo1bR2 are outer primers which replace the first nucleic acid obtained with Mo1bHF and Mo1bHR, respectively, as synthesis origins. Since the outer primer after Mo1bHF (or Mo1bHR) synthesis is the primer for the complementary strand synthesis origin. These are designed to anneal into the ring-like regions by exploiting the proximity stacking phenomenon. In addition, setting these primers to high concentrations allows annealing of Mo1bHF (or Mo1bHR) to occur preferentially.
The nucleotide sequence constituting each primer is shown in the sequence table, and the structural features of the primers are summarized below. In addition, the positional relationship for each region of the target nucleotide sequence is shown in FIG. 5.
The combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers are shown in Table 1 below.
TABLE 1 primer and oligonucleotide information
| Primer and method for producing the same | 5 '-side region/3' -side region |
| Mo1bHF | Identical/reverse to the Flc region in the complementary strand synthesized by Mo1bHF, R1 region in MERS-orf1b |
| Mo1bHR | Same as/reverse to the F1 region in MERS-orf1b in the Rlc region in the complementary strand synthesized by Mo1bHB |
| Mo1bF2 | Complementary to F2c adjacent to the 3' -side of the F1c region in MERS-orf1b |
| Mo1bR2 | Complementary to R2c adjacent to the 5' -side of the R1 region in MERS-orf1b |
By the primer, nucleic acids in which R1 and R1rc are complementary and F1c and F1R are complementary to each other to form a helical loop are synthesized. Combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers are shown below.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
Primer:
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
target MERS-orf1b dsDNA/SEQ ID NO.5
The specific nucleotide sequences are as follows:
SEQ ID NO.l：GTACGAAGGGCATTACGCTCTCGTGTTATTTCCAGG；
SEQ ID NO.2：GGACCTTTATTGTGCTCTCGCATTACGGGAAGCATG；
SEQ ID NO.3：TACCCGCAAATGTCCCATA；
SEQ ID NO.4：TGTAGAGGCACATTGGTG；
SEQ ID NO.5：
AAGACGAGTGATGAGCTTTGCGTGAATCTTAATTTACCCGCAAATGTCCCATACTCTCGTGTTATTTCCAGGATGGGCTTTAAACTCGATGCAACAGTTCCTGGATATCCTAAGCTTTTCATTACTCGTGAAGAGGCTGTAAGGCAAGTTCGAAGCTGGATAGGCTTCGATGTTGAGGGTGCTCATGCTTCCCGTAATGCATGTGGCACCAATGTGCCTCTACAA。
the mixture was reacted at 63 ℃ for 1 hour, after which the reaction was terminated at 80 ℃ for 10 minutes and then transferred again to ice-precooled water.
Confirmation of the reaction: mu.L of the loading buffer was added hydraulically to 5. mu.L of the above reaction solution, and the sample was electrophoresed for 1 hour on 90mV 1% agarose gel (TAE lysis) prestained in GelRed (Biotum). The Byunnan O0107DNA Ladder is used as a molecular weight marker. Gel after electrophoresis to verify nucleic acids. The results are shown in FIG. 6, with respect to the lower sample for each lane.
1. Molecular weight marker DNA ladder
2. 1fmolM13mpl8 dsDNA。
Example 2 confirmation of the reaction product by digestion with restriction enzymes
In order to clarify the nucleic acid structure obtained in example 1 of the present invention having complementary nucleotide sequences linked in a circular structure within a single strand, the product was digested with restriction enzymes. If a theoretical fragment can be generated by digestion, while the absence (dispear) of high molecular weight as observed in example 1 produces an unclear striped pattern and bands that are not electrophoresed, any of these products would be expected to be a nucleic acid of the invention with complementary sequences alternately ligated into the single strand.
The reaction solution in example 1 was precipitated and purified by treatment with phenol and precipitation with ethanol, the resulting precipitate was recovered and redissolved in ultrapure water, digested with restriction enzyme HindIII at 37 ℃ for 2 hours, and the sample was electrophoresed for 1 hour on a 90mV GelRed prestained (Biotum) 1% agarose gel (TAE lysis). The Byunnan O0107DNA Ladder is used as a molecular weight marker. Gel after electrophoresis to verify nucleic acids. The results are shown in FIG. 7, with respect to the lower sample for each lane.
1. Molecular weight marker DNA ladder
2. 1fmolM13mpl8 dsDNA
3 HindIII digestion of the purified product
Example 3 validation of reaction products using EvaGreen
Like SYBR Green I, EvaGreen is a dye with Green excitation wavelength and combined with all the dsDNA double helix minor groove regions, and the inhibition of the dye on nucleic acid amplification reactions such as PCR is far smaller than that of the dye. In the free state, EvaGreen emits weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of EvaGreen is correlated with the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected from the fluorescence signal.
Combinations of reaction solutions for the method of synthesizing the nucleic acid of the present invention by these primers are shown below.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
120μM
Primer:
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
target MERS-orf1b dsDNA/SEQ ID NO.5
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 75 min. The fluorescence intensity curve with respect to the reaction time is shown in FIG. 8. The fluorescence detection is applied to the target that real-time monitoring can be realized, and the result can be judged in advance through a real-time amplification curve.
Example 4 comparison of target Gene amplification at different concentrations Using EvaGreen-based real-time fluorescence
Reaction solution combination (25. mu.L) eight tubes
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
Primer:
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
target MERS-orf1b dsDNA/SEQ ID NO.5
8 tubes were set for the same reaction solution, and the difference between the addition of only targets was 108Copies，107Copies，106Copies，105Copies，104Copies，103Copies，102Copies, pure water (0 Copies).
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 90 min. The fluorescence intensity of the amplification system at different target concentrations as a function of reaction time is shown in FIG. 9. The result shows that the linear relation of the results obtained by the method is better, and the method can be applied to quantitative detection of the target nucleic acid.
Example 5 comparison of RNA target Gene amplification at different concentrations Using real-time EvaGreen-based fluorescence
The AMV reverse transcriptase can synthesize cDNA by taking RNA as a template, and the detection of the RNA can be realized by matching with Bst DNA polymerase.
Reaction solution combination (25. mu.L)
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
10U AMV reverse transcriptase
1X EvaGreen(Biotum)
Primer:
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
target MERS-orf1 bssiRNA/SEQ ID NO.5
8 tubes were set for the same reaction solution, and only the addition of the target was distinguished, 10 each8Copies，107Copies，106Copies，105Copies，104Copies，103Copies，102Copies, pure water (0 Copies).
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 90 min. The fluorescence intensity of the amplification system with different target concentrations as a function of reaction time is shown in FIG. 10. This result demonstrates that the method is equally applicable to RNA detection.
Example 6 amplification of target genes at different concentrations Using Fin
Reaction solution combination (25. mu.L) eight tubes
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M betaine
1.25mM dNTP
8U Bst DNA polymerase (NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
Primer:
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
800nM Mo1bFin/SEQ ID NO.6
800nM Mo1bFin/SEQ ID NO.7
target MERS-orf1b dsDNA/SEQ ID NO.5
The specific nucleotide sequences of the Fin and Rin primers are as follows:
SEQ ID NO.6：ACAGTTCCTGGATATCCTAAGCT；
SEQ ID NO.7：ACAGCCTCTTCACGAGTAATG。
8 tubes were set for the same reaction solution, and only the addition of the target was distinguished, 10 each6Copies，105Copies，104Copies，103Copies，102Copies，101Copies，100Copies, pure water (0 Copies).
The ABI StepOne real time PCR reaction temperature is set to be 63 ℃ constantly, and the reaction time is set to be 75 min. The fluorescence intensity of the amplification system at different target concentrations as a function of reaction time is shown in FIG. 11. Comparison of this result with that of example 6 shows that the addition of Fin oligonucleotide significantly reduces the amplification time.
The present invention may be embodied in many different forms and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
<110> institute of Process engineering of Chinese academy of sciences
<120> a method for synthesizing nucleic acid at constant temperature
<160>7
<210>1
<211>36
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>1
gtacgaaggg cattacgctc tcgtgttatt tccagg 36
<210>2
<211>36
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>2
ggacctttat tgtgctctcg cattacggga agcatg 36
<210>3
<211>19
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>3
tacccgcaaa tgtcccata 19
<210>4
<211>18
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>4
tgtagaggca cattggtg 18
<210>5
<211>225
<212>DNA
<213> Artificial sequence
<220>
<223>MERS-orf1b
<400>5
aagacgagtg atgagctttg cgtgaatctt aatttacccg caaatgtccc atactctcgt 60
gttatttcca ggatgggctt taaactcgat gcaacagttc ctggatatcc taagcttttc 120
attactcgtg aagaggctgt aaggcaagtt cgaagctgga taggcttcga tgttgagggt 180
gctcatgctt cccgtaatgc atgtggcacc aatgtgcctc tacaa 225
<210>6
<211>23
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>6
acagttcctg gatatcctaa gct 23
<210>7
<211>21
<212>DNA
<213> Artificial sequence
<220>
<223> primer
<400>7
acagcctctt cacgagtaat g 21
Claims (10)
1. A method for synthesizing nucleic acid under isothermal conditions, comprising the steps of:
1) providing a nucleic acid having a F1r region at the 3' end that anneals to a F1c region on the same strand and forming a helical loop by simultaneous annealing of the F1r region to a Flc region;
2) synthesizing a complementary strand of the nucleic acid of the step 1) by using the nucleic acid as a template, wherein the R1rc region of the complementary strand is annealed with the R1 region, and the 3' end of the F1R region annealed by the Flc region forms a spiral loop and is used as a synthesis starting point;
3) complementary strand synthesis is performed by polymerase-catalyzed strand displacement-type complementary strand synthesis reaction to displace the complementary strand synthesized in step 2), wherein the polynucleotide comprises at its 3' end a sequence complementary to any region of the complementary strand synthesized in step 2).
2. The method for synthesizing nucleic acid according to claim 1, wherein the method for preparing nucleic acid according to step 1) comprises the steps of:
i) an annealing step of annealing a first oligonucleotide I to a F1c region of a template, wherein the 3' end of the template comprises a F1c region and a F2c region located 3' to the F1c region and the 5 ' end of the template comprises a R1 region and a R2 region located 5 ' to the R1 region, wherein said first oligonucleotide I, comprising a R1R region and a Fl region, said Rlr region is linked to the 5 ' to the F1 region, wherein,
region F1: a region having a nucleotide sequence complementary to the region of the template F1c,
R1R region: a region opposite to the region of template R1;
ii) a step of synthesizing a first nucleic acid having a nucleotide sequence complementary to the template, the step synthesizing the first nucleic acid with the F2 region of the first oligonucleotide I as a starting point of synthesis, the 3' -end of the first nucleic acid having an F1 region annealing to the partial F1c region on the same strand, and a helical loop formed by simultaneous annealing of the F1 region and the Flc region;
iii) displacing the first nucleic acid synthesized in step ii) with a polymerase-catalyzed strand displacement reaction in which the first outer primer I annealing to the F2c region 3' to F1c in the template serves as the start of synthesis, and
iv) an annealing step of annealing a second oligonucleotide II to the R1c region of the first nucleic acid obtained in step iii), wherein the second oligonucleotide II comprises a R1 region and a F1R region, and a Flr region is linked to the 5' side of the R1 region, the second oligonucleotide II being the reverse sequence of the first oligonucleotide I; wherein,
region R1: a region having a nucleotide sequence complementary to the R1c region of the first nucleic acid,
flr area: a region that is inverted with respect to the F1 region of the first nucleic acid;
v) synthesizing a second nucleic acid with the second oligonucleotide II as the synthesis starting point, and replacing the second nucleic acid by a polymerase-catalyzed strand displacement reaction to obtain the nucleic acid of step 1); wherein the second outer primer II annealing to the R2c region 3' to R1c in the first nucleic acid serves as the origin of synthesis when the second nucleic acid is replaced.
3. The method for synthesizing nucleic acid according to claim 2, wherein the template in step i) is RNA and the first nucleic acid in step ii) is synthesized by an enzyme having reverse transcriptase activity.
4. The method for synthesizing a nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid synthesized in step 3) is a nucleic acid having a nucleotide sequence complementary to each other end to end on one strand thereof.
5. A method for synthesising a nucleic acid as claimed in any one of claims 1 to 3 wherein the following relationship exists for the melting temperature of each oligonucleotide with its complementary region in the template: (outer primer or template 3' side region) of ≦ (F2c or F2, and, R2c or R2) ≦ (F1c or F1, and, Rlc or Rl).
6. The method for synthesizing nucleic acid according to claim 2, wherein the second nucleic acid obtained is accelerated in nucleic acid amplification by introducing an acceleration probe Xin, wherein Xin is located in the middle region from the F1 region to the R1 region.
7. The method for synthesizing a nucleic acid according to any one of claims 1 to 3, wherein the steps 1) to 3) are repeated using the replaced complementary strand of step 3) as a template.
8. A kit for synthesizing a nucleic acid under isothermal conditions, the kit comprising:
an oligonucleotide I comprising a F1 region and a R1R region, said R1R region being linked to the 5' side of the F1 region, wherein,
region F1: a region having a nucleotide sequence complementary to the F1c region of the template, and
R1R region: a region opposite to the region R1 of the template;
an oligonucleotide II comprising a region R1 and a region F1R, said region F1R being linked to the 5' side of the region R1, wherein,
region R1: a region having a nucleotide sequence complementary to the R1c region of the template, and
f1 r: a region opposite to the F1 region of the template;
a first primer I having a nucleotide sequence complementary to the F2c region 3' to the F1c region in the nucleic acid as a template;
a second primer II having a nucleotide sequence complementary to the R2c region 3' to the R1c region in the nucleic acid as a template;
a DNA polymerase catalyzing a strand displacement-type complementary strand synthesis reaction, and,
a nucleotide that serves as a substrate for the DNA polymerase.
9. The kit of claim 10, further comprising an acceleration probe Xin, wherein Xin is a nucleotide fragment located in the middle segment from the F1 region to the R1 region of the template.
10. Use of a kit according to claim 8 or 9 for synthesizing a nucleic acid or detecting a target nucleotide sequence in a sample.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107446919A (en) * | 2017-09-14 | 2017-12-08 | 中科芯瑞(苏州)生物科技有限公司 | The method and kit of nucleic acid under a kind of constant temperature |
| WO2019051732A1 (en) * | 2017-09-14 | 2019-03-21 | 中科芯瑞(苏州)生物科技有限公司 | Method and kit for synthesizing nucleic acid under constant temperature conditions |
| CN113201583A (en) * | 2021-04-29 | 2021-08-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
| CN113322338A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院大学宁波生命与健康产业研究院 | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit |
| CN114317689A (en) * | 2022-02-07 | 2022-04-12 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition for non-diagnosis purpose, kit and application |
| WO2022077822A1 (en) * | 2020-10-15 | 2022-04-21 | 德歌生物技术(山东)有限公司 | Method for designing primers, primers, and method for isothermal amplification of nucleic acid fragments |
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| CN107446919A (en) * | 2017-09-14 | 2017-12-08 | 中科芯瑞(苏州)生物科技有限公司 | The method and kit of nucleic acid under a kind of constant temperature |
| WO2019051732A1 (en) * | 2017-09-14 | 2019-03-21 | 中科芯瑞(苏州)生物科技有限公司 | Method and kit for synthesizing nucleic acid under constant temperature conditions |
| CN107446919B (en) * | 2017-09-14 | 2020-04-28 | 中科芯瑞(苏州)生物科技有限公司 | Method and kit for synthesizing nucleic acid under constant temperature condition |
| WO2022077822A1 (en) * | 2020-10-15 | 2022-04-21 | 德歌生物技术(山东)有限公司 | Method for designing primers, primers, and method for isothermal amplification of nucleic acid fragments |
| CN113201583A (en) * | 2021-04-29 | 2021-08-03 | 中国科学院大学宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
| CN113201583B (en) * | 2021-04-29 | 2022-02-08 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition, kit and application |
| CN113322338A (en) * | 2021-07-19 | 2021-08-31 | 中国科学院大学宁波生命与健康产业研究院 | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit |
| CN113322338B (en) * | 2021-07-19 | 2022-03-22 | 国科宁波生命与健康产业研究院 | CDA primer group and kit for detecting Shigella and application of CDA primer group and kit |
| CN114317689A (en) * | 2022-02-07 | 2022-04-12 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition for non-diagnosis purpose, kit and application |
| CN114317689B (en) * | 2022-02-07 | 2024-04-09 | 国科宁波生命与健康产业研究院 | Method for synthesizing nucleic acid under constant temperature condition for non-diagnostic purpose, kit and application |
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