WO2019051732A1 - 一种恒温条件下合成核酸的方法及试剂盒 - Google Patents

一种恒温条件下合成核酸的方法及试剂盒 Download PDF

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WO2019051732A1
WO2019051732A1 PCT/CN2017/101789 CN2017101789W WO2019051732A1 WO 2019051732 A1 WO2019051732 A1 WO 2019051732A1 CN 2017101789 W CN2017101789 W CN 2017101789W WO 2019051732 A1 WO2019051732 A1 WO 2019051732A1
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region
nucleic acid
complementary
strand
synthesis
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PCT/CN2017/101789
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English (en)
French (fr)
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杜昱光
毛瑞
王倬
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中科芯瑞(苏州)生物科技有限公司
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Priority to EP20173944.8A priority Critical patent/EP3808843A1/en
Priority to EP17905910.0A priority patent/EP3476938B1/en
Priority to PCT/CN2017/101789 priority patent/WO2019051732A1/zh
Publication of WO2019051732A1 publication Critical patent/WO2019051732A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

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  • the present invention relates to the field of genetic engineering technology, and in particular, to a method for synthesizing nucleic acid under constant temperature conditions, in particular to a synthetic method for forming a special structural nucleic acid composed of a specific nucleotide sequence, and based on the specific nucleic acid A useful method of amplifying nucleic acids in a sequence.
  • the exponential amplification result of this method makes it highly sensitive, and has established its position in the field of molecular biological method detection. After several decades of development, it has a series of mature products. In addition, amplification products are recyclable and are therefore widely used as an important tool to support genetic engineering techniques such as gene cloning and structural determination.
  • the PCR method clearly has the following problems: in the actual operation, a special program temperature control system must be used; 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 false positive problem is more prominent.
  • NASBA nucleic acid sequence-dependent amplification
  • RCA rolling circle amplification
  • SDA strand displacement amplification
  • LAMP loop-mediated isothermal amplification
  • HDA helicase-dependent amplification
  • RPA recombinant polymerase amplification
  • NASBA also known as TMA (Turning-Mediated Amplification Method)
  • TMA Traning-Mediated Amplification Method
  • the method uses DNA polymerase to synthesize a target RNA as a template and a probe linked to a T7 promoter, and the second probe enters a double strand to generate a product, and then uses the generated double-stranded DNA as a template to pass T7 RNA polymerase.
  • a large amount of RNA product is amplified by transcription.
  • NASBA requires a heat denaturation step until double stranded DNA is formed, but the subsequent transcription reaction is carried out by isothermal conditions by T7 RNA polymerase.
  • RCA Rolling Circle Amplification
  • the primers combined with the template can only achieve circular nucleic acids in the original method.
  • Amplification In order to make the method universal for linear DNA amplification, a single-stranded DNA complementary to a padlock probe or a circular probe is shown to have a series of nucleotide sequences with a padlock probe or a circular probe.
  • Complementary single-stranded DNA can be synthesized continuously in the presence of target nucleotides (Lizardi, Huang et al. 1998). This method also has the problem of requiring multiple enzymes.
  • the initiation of complementary strand synthesis depends on the reaction linking the two adjacent regions, and its specificity is substantially the same as in the 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 uses a specific DNA polymerase to synthesize a complementary strand starting from the 3'-side complementary primer of the nucleotide sequence of interest to replace the double-stranded 5'-side sequence. Since the newly synthesized complementary strand replaces the 5'-side duplex, the technique is referred to as the SDA method.
  • the restriction enzyme recognition sequence is inserted as a primer into the annealing sequence to remove the temperature change step necessary in the PCR method.
  • the 3'-OH group is supplied as a synthetic starting point of the complementary strand by restriction enzyme-generated nicks, and the first synthesized complementary strand is released by strand displacement synthesis to release a single strand, and then used again as a template for the following complementary strand synthesis.
  • SDA amplification products differ from natural nucleic acid structures and have limitations on the use of restriction enzymes to cleave or apply amplification products to gene cloning. This is also the main reason for the high cost.
  • the nucleotide sequence of the same restriction enzyme recognition sequence for introducing a gap may exist in the region to be synthesized, thus possibly preventing the synthesis of the fully complementary strand.
  • HSA Helicase-dependent Isothermal DNA Amplification
  • Vincent, Xu et al. 2004 This technique mimics the natural process of DNA replication in nature, using a helicase to unwind DNA double-strands under constant temperature conditions, while a single-stranded DNA-binding protein (SSB) is used to stabilize the unfolded single.
  • SSB single-stranded DNA-binding protein
  • the strands are provided with a binding template for the primers, which are then catalyzed by a DNA polymerase to synthesize a complementary strand.
  • the newly synthesized double strand is decomposed into a single strand under the action of a helicase, and enters the above-mentioned cyclic amplification reaction as a template for the next round of synthesis, and finally achieves an exponential growth of the target sequence.
  • RPA Recombinase Polymerase Amplification
  • the core of LAMP technology (Notomi, Okayama et al. 2000) is to use four high-activity strands to replace DNA polymerases by designing four specific primers for six regions on the target gene, so that strand-replacement DNA synthesis is constantly self-circulating. .
  • the technique can achieve 109-1010 times amplification in 15-60 minutes, and the reaction can generate a large amount of amplification product, that is, white precipitate of magnesium pyrophosphate, and the presence or absence of the white precipitate can be visually observed to determine whether the target gene exists.
  • Rongyan has also developed a turbidity meter to achieve real-time monitoring of the amplification reaction. In addition to high specificity and high sensitivity, the LAMP method is very simple to operate.
  • 5S rRNA is highly conserved and is often chosen as a marker for molecular detection. Since the nucleic acid sequence of 5S rRNA is short, the detection of this marker is now generally carried out by PCR. Isothermal amplification of nucleic acids has the advantage of being simple and quick, but it is difficult to apply to the detection of 5S rRNA.
  • LAMP primers are composed of F3, B3, FIP and BIP. The nucleic acid sequence required for designing these primers requires about 120 bases, not to mention between F2 and B2. The interval is difficult to meet the harsh conditions of the LAMP primer design, so it cannot be applied to the detection of 5S rRNA.
  • the object of the present invention is to provide a method for synthesizing nucleic acid, which is inspired by the common double helix structure of DNA, molecular beacon probe and LAMP, and redesigns and plans the working mode of primers based on PCR primers to form competitive stems.
  • the ring initiates the amplification of the structure, the formation of the structural loop is designed, and features the optional use of external primers. More specifically, it provides a novel low cost method for efficiently synthesizing nucleic acids by means of sequences. That is, it is an object of the present invention to provide a method for accomplishing nucleic acid synthesis and amplification by a single enzyme and isothermal conditions.
  • Another object of the present invention is to provide a method for synthesizing nucleic acid, which is capable of rapidly synthesizing a highly specific nucleic acid which is difficult to achieve by modifying an existing nucleic acid synthesis reaction principle, and a method for amplifying a nucleic acid by the synthetic method .
  • One advantage of the present invention is that rapid amplification of the gene can be achieved using a single enzyme thermostat system for short-chain nucleic acid fragments (ideal fragments can be only 60 bp, shorter than the ideal fragment of LAMP minimum 120 bp).
  • the present invention utilizes a polymerase-catalyzed strand displacement type of complementary strand synthesis without complicated temperature control, which is beneficial to the synthesis of nucleic acids.
  • the DNA polymerase is an enzyme used in methods such as SDA, RCA, and LAMP.
  • the present inventors have improved the supply of the known method 3'-OH, and as a result, it has been found that by using an oligonucleotide having a specific structure, a 3'-OH structure can be provided without any additional enzyme reaction, thereby obtaining the present invention.
  • the present invention relates to a method of synthesizing a nucleic acid, a method of amplifying a nucleic acid by the nucleic acid synthesis method, and a kit for synthesizing a nucleic acid using the method.
  • the invention provides a method for synthesizing nucleic acid under constant temperature conditions, comprising the following steps:
  • nucleic acid having a N' region at the 5' end of the nucleic acid which can anneal to the N region on the same chain, and having a Nc region at the 3' end of the nucleic acid which can anneal to the N region on the same chain
  • the Nc region of the 5' end and the 3' end of the nucleic acid has a competitive relationship with the annealing of the N region on the same chain;
  • step 2) using the nucleic acid of step 1) as a template, the 3' end of the Nc region which has been annealed to the N region is a synthetic starting point, and synthesizes its own complementary strand;
  • Complementary strand synthesis is carried out by polymerase catalytic chain displacement type complementary strand synthesis reaction to replace the complementary strand synthesized in step 2).
  • the Nc region of the 'end and 3' ends has a competitive relationship with the annealing of the N region on the same chain;
  • An oligonucleotide I comprises an N region and an F1 region;
  • nucleic acid A synthesizing its own complementary strand using the nucleic acid provided in step 1) as a template, and the 3' end of the Nc region which has been annealed to the N region is a synthetic starting point; the nucleic acid sequence after the synthesis is referred to as nucleic acid A;
  • the second oligonucleotide II includes an R1 region and an Nc region;
  • Fig. 1 there is shown a step diagram corresponding to the above synthetic nucleic acid in the present invention.
  • the method for preparing the nucleic acid of the above step 1) comprises the following steps:
  • F1 region a region having a nucleotide sequence complementary to the F1c region
  • N region a region of a nucleotide sequence complementary to the Nc region
  • 1-2 synthesizing a first nucleic acid using the F1 region of the first oligonucleotide I as a synthesis starting point; the first nucleic acid having a nucleotide sequence complementary to the template, 5 of the first nucleic acid
  • the end has an N region that can anneal to the Nc region on the same chain, and the stem ring can be formed by annealing the Nc region and the N region;
  • step 1-2 using a polymerase catalytic chain displacement reaction to obtain the first nucleic acid synthesized in step 1-2);
  • R1 region a region having a nucleotide sequence complementary to the R1c region
  • Nc region a region of a nucleotide sequence complementary to the N region
  • step 1 a step corresponding to the nucleic acid described in the above second nucleic acid (i.e., step 1).
  • the template in step 1-1) is RNA
  • the first nucleic acid in step 1-2) is synthesized by an enzyme having reverse transcriptase activity.
  • the present invention is applicable to various DNAs and RNAs, such as DNA and RNA of various animal and plant cells, bacteria and viruses, and the like.
  • DNAs and RNAs such as DNA and RNA of various animal and plant cells, bacteria and viruses, and the like.
  • cDNA and RNA detection of H1 gene and N1 gene for H1N1 virus cDNA and RNA detection for MERS-CoV virus, such as: orf1a, orf1b segment of the RNA; DNA for herpes simplex virus type III Detection, etc.
  • the nucleic acid fragments of the F1c region, the N region and the R1 region are both 15-60 bp. Further preferably, the nucleic acid fragments of the F1c region, the N region and the R1 region are both 20 bp.
  • the method for synthesizing nucleic acid under the constant temperature condition further comprises: the nucleic acid strand obtained in the step 5) is capable of autonomous pairing infinite extension, and the 3' Nc region on the nucleic acid strand is paired with the complementary segment N region on the chain.
  • the nucleic acid strand is continuously extended as a starting point of synthesis using itself as a template.
  • a method of synthesizing a nucleic acid according to the present invention wherein the synthetic nucleic acid is A nucleic acid having a first-tail complementary nucleotide sequence on one strand.
  • the method of synthesizing a nucleic acid according to the present invention wherein the constant temperature means that the entire reaction process is carried out at a temperature ranging from 60 to 65 °C.
  • nucleic acid amplification is accelerated by introducing a method of accelerating the primers X2 and/or Xin; wherein X2 is located on the 5' side of the F1 region and the R1 region of the complementary strand of the original nucleic acid.
  • the section, Xin is located in the middle section of the F1c zone to the N zone and the Nc zone to R1c.
  • the polymerase used in the polymerase catalytic chain displacement reaction of the present invention is 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), ⁇ 29phage
  • Bst DNA polymerase or Bca (exo-) DNA polymerase is preferably used.
  • a method of synthesizing a nucleic acid according to the present invention wherein a melting temperature adjusting agent can be added to the polymerase catalytic chain displacement reaction.
  • the melting temperature adjusting agent is preferably a betaine, and further preferably, the concentration of the betaine in the reaction solution is 0.2 to 3.0M.
  • kit for synthesizing nucleic acid under constant temperature conditions of the invention characterized in that the kit comprises:
  • a first oligonucleotide I comprising an F1 region and an N region, the N region being linked to the 5' side of the F1 region, wherein
  • F1 region a region having a nucleotide sequence complementary to the F1c region
  • N region a region of a nucleotide sequence complementary to the Nc region
  • a second oligonucleotide II comprising an R1 region and an Nc region, wherein the Nc region is linked to the 5' side of the R1 region, wherein
  • R1 region a region having a nucleotide sequence complementary to the R1c region
  • Nc region a region of a nucleotide sequence complementary to the N region
  • Nucleotide which serves as a substrate for the DNA polymerase.
  • kit according to the present invention, wherein the kit further comprises a nucleic acid for detecting A reagent for detecting a reaction product.
  • kit further comprises an acceleration primer X2 and/or Xin, wherein X2 is a segment located on the 5' side of the F1 region and the R1 region of the complementary strand of the original nucleic acid, and Xin is located F1c zone to N zone and Nc zone to the middle section of R1c.
  • X2 is a segment located on the 5' side of the F1 region and the R1 region of the complementary strand of the original nucleic acid
  • Xin is located F1c zone to N zone and Nc zone to the middle section of R1c.
  • the DNA polymerase is Bst DNA polymerase, Bca (exo-) DNA polymerase, DNA polymerase I Klenow fragment, Vent DNA polymerase, Vent (Exo-) One or more of DNA polymerase, Deep Vent DNA polymerase, Deep Vent (Exo-) DNA polymerase, ⁇ 29phage DNA polymerase, and MS-2phage DNA polymerase.
  • Bst DNA polymerase or Bca (exo-) DNA polymerase is preferably used.
  • the invention provides the use of the above kit for synthesizing nucleic acids or detecting target nucleotide sequences in a sample.
  • the method for synthesizing a nucleic acid according to the present invention provides a method for detecting a target nucleotide sequence in a sample, comprising performing amplification by the method for synthesizing a nucleic acid of the present invention using a target nucleotide as a template, and observing whether or not amplification is generated product.
  • a probe comprising a nucleotide sequence complementary to the formed stem-loop structure was added to the above amplification product, and the hybridization between the two was observed. It is also possible to label the probe on the particles and observe the aggregation reaction occurring by hybridization.
  • the amplification method can be carried out in the presence of a nucleic acid detection reagent, and it is observed whether or not an amplification product is generated based on a signal change of the detection reagent.
  • the method for synthesizing a nucleic acid according to the present invention may further provide a method for detecting a mutation of a target nucleotide sequence in a sample, comprising performing amplification by a method for synthesizing a nucleic acid according to the present invention using a target nucleotide as a template.
  • the mutation in the nucleotide sequence as a target of amplification hinders the synthesis of any complementary strand constituting the amplification method, thereby detecting the mutation.
  • a single-stranded nucleic acid which can form a nucleotide sequence of a competitive stem-loop structure which is a nucleic acid having a target nucleic acid sequence which is alternately linked in a single strand in a certain order with a complementary nucleic acid sequence.
  • the nucleic acids synthesized by the present invention consist essentially of mutually complementary strands joined by a stem-and-loop structure. Referring to Figure 5, a schematic representation of an ideal amplified nucleic acid product formed by the synthetic method of the present invention.
  • a chain that can be separated into two or more molecules is called a single strand.
  • the complementary nucleotide sequence in the same strand can form base pairing, and the present invention can obtain the product of intramolecular base pairing by allowing base pairing of nucleic acids having a nucleotide sequence end-to-end linked in a single strand in the same strand.
  • the product contains a region that constitutes a distinct double strand and a loop that does not involve base pairing.
  • the nucleic acid of the present invention having a nucleotide sequence of a competitive stem-loop structure can be defined as a single-stranded nucleic acid comprising a complementary nucleotide sequence which can anneal in the same strand.
  • a nucleotide having a complementary nucleotide sequence can be annealed to a loop that does not involve base pairing.
  • the loop-forming sequence can be any nucleotide sequence.
  • the loop-forming sequences are capable of base pairing to initiate synthesis of the complementary strand for substitution. Sequences different from the nucleotide sequences located in other regions are preferentially provided to obtain specific annealing.
  • the substantially identical nucleotide sequence in the present invention is defined as follows: when a complementary strand synthesized by using a sequence as a template anneals to a target nucleotide sequence to supply a starting point for synthesizing a complementary strand, the sequence is substantially identical to the target nucleotide sequence the same.
  • a sequence substantially identical to F1 includes not only the same sequence as F1 but also a nucleotide sequence that can serve as a template, which can give a nucleotide sequence annealed to F1 and can serve as a complementary strand. starting point.
  • annealing in the present invention refers to the formation of a nucleic acid of complementary structure by base pairing according to Watson-Crick's law.
  • annealing may occur if the complementary nucleotide sequences in the molecule are base-paired.
  • the base-paired nucleic acid constitutes a double-stranded structure, so that the meanings expressed by annealing and hybridization of the present invention have overlapping portions.
  • the nucleotide sequence of the constituent nucleic acids of the invention is at least one.
  • the nucleotide sequence logarithm can be an integral multiple of one.
  • the complementary nucleotide sequence of the constituent nucleotides of the present invention has no theoretical upper limit.
  • the synthetic product nucleic acid of the present invention consists of a plurality of sets of complementary nucleotide sequences, the nucleic acid is repeated by the same nucleotide. Sequence composition.
  • the single-stranded nucleotide of the nucleotide sequence having a competitive stem-loop structure synthesized by the present invention has a different structure from a naturally occurring nucleic acid, and is generally known to be derivatized with a nucleotide when the nucleic acid is synthesized by a nucleic acid polymerase.
  • a nucleic acid derivative can be synthesized.
  • Nucleotide derivatives used include radioisotope labeled nucleotides or nucleotide derivatives that bind to a ligand tag such as biotin or digoxin. These nucleotide derivatives can be used to label product nucleic acids.
  • the substrate is a fluorescent nucleotide
  • the product nucleic acid may be a fluorescent derivative.
  • the synthesis of a nucleic acid having the above structure can be initiated by DNA polymerase having a strand displacement activity and the Nc region having a portion N region at the 3'-end and the same strand to anneal to a complementary strand.
  • DNA polymerase having a strand displacement activity
  • the Nc region having a portion N region at the 3'-end and the same strand to anneal to a complementary strand.
  • the present invention provides a competitive hairpin loop.
  • nucleic acid is used in the present invention, and the nucleic acid of the present invention generally includes both DNA and RNA.
  • the template for synthesizing nucleic acids of the present invention nucleic acids or modified nucleotides derived from natural DNA or RNA whose nucleotides are replaced by artificial derivatives are also included in the nucleic acid range of the present invention.
  • the nucleic acids of the invention are included in biological samples, including tissues, cells, cultures and secretions of animals, plants or microorganisms, and extracts thereof.
  • Biological samples of the invention include intracellular parasite genomic DNA or RNA, such as viruses or mycoplasmas.
  • the nucleic acids of the invention are typically derived from a nucleic acid contained in the biological sample.
  • a nucleic acid which is synthesized from mRNA and which is amplified based on a nucleic acid derived from a biological sample is a typical example of the nucleic acid of the present invention.
  • the nucleic acid of the present invention is characterized in that the Nc region is provided at the 3'- and 5'-ends, and can be annealed with a partial N region on the same chain, and the Nc region is annealed with the N region on the same chain to form a hairpin ring.
  • the nucleic acid is obtained in various methods, and the Nc region of the 5' end and the 3' end of the nucleic acid competes with the annealing of the N region on the same chain. Furthermore, it is only possible to extend under the action of DNA polymerase when the Nc region at the 3'-end is annealed to the N region.
  • FIG. 3 there is shown a schematic diagram of a competitive hairpin structure formed by a single-stranded nucleic acid of the present invention and subsequent amplification.
  • Figure 4 is a schematic diagram of a subsequent amplification reaction of nucleic acids.
  • NF in FIGS. 3 and 4 means the first oligonucleotide I
  • NR in FIG. 4 means the second oligonucleotide II.
  • nucleotide sequence features of the oligonucleotides of the present invention are not meant to be absolutely identical and absolutely complementary. That is, a sequence identical to a sequence includes a sequence complementary to a nucleotide sequence annealed to a sequence. Complementary order The column is a sequence that can be annealed under stringent conditions, providing the 3'-end as the starting point for the synthesis of the complementary strand.
  • an oligonucleotide is a nucleotide that satisfies two requirements, i.e., must be capable of forming a complementary base pairing, and supplying an -OH group at the 3'-end is a starting point for complementary strand synthesis. Therefore, its main chain is not necessarily limited to a linkage of a phosphodiester bond. For example, it may be composed of a phosphorothioate derivative or a peptide-based peptide nucleic acid, and the phosphorothioate derivative is S-substituted O. Bases are those bases that are complementary to each other.
  • the oligonucleotide of the present invention can be used not only as a starting point for synthesis but also as a template for complementary strand synthesis.
  • the term polynucleotide of the invention includes oligonucleotides.
  • polynucleotide as used herein has a chain length that is not limited, and the term “oligonucleotide” as used herein refers to a polymer of nucleotides having a relatively short chain length.
  • the oligonucleotide strand of the present invention has such a length that it can base pair with the complementary strand and maintain a certain specificity. Specifically, it consists of 5 to 200 bases, more preferably 10 to 50 base pairs.
  • the known polymerase is identified to have a chain length of at least 5 bases. The polymerase catalyzes a nucleic acid synthesis reaction that relies on the sequence. Therefore, the chain length of the annealed portion should be longer than this length. In addition, it is statistically desirable to lengthen 10 bases or longer to obtain target nucleotide specificity. On the other hand, it is difficult to prepare a nucleotide sequence too long by chemical synthesis.
  • the above chain length is an example of a desired range.
  • the chain length of the illustration refers to the chain length of the portion that anneals to the complementary strand.
  • the oligonucleotides of the invention may eventually anneal at least to the two regions, respectively.
  • the chain length exemplified herein is understood to be the chain length of each region that makes up the oligonucleotide.
  • the oligonucleotides of the invention may be labeled with known labels.
  • Labels include binding ligands such as digoxin and biotin, enzymes, fluorescents, illuminants, and radioisotopes.
  • a technique for replacing a base constituting an oligonucleotide by a fluorescent analog is well known (W095/05391, Proc. Natl. Acad. Sci. USA, 91, 6644-6648, 1994).
  • oligonucleotides of the invention may also be incorporated into a solid phase.
  • any portion of the oligonucleotide may be labeled with a binding ligand, such as biotin, indirectly by a binding ligand such as immobilized avidin.
  • a binding ligand such as biotin
  • immobilized avidin When the immobilized oligonucleotide is the starting point of synthesis, the nucleic acid of the synthetic reaction product is captured by the solid phase, which will facilitate its separation.
  • nucleic acid-specific indicators or hybrids with labeled probes The separation can be detected.
  • the nucleic acid product obtained by the present method is recovered for a target nucleic acid fragment in which the target nucleic acid fragment can be digested by a restriction enzyme.
  • template refers to a nucleic acid used as a template for the synthesis of a complementary strand.
  • a complementary strand having a nucleotide sequence complementary to a template means a strand corresponding to the template. But the relationship between the two is only relative. That is, the synthesized complementary strand can once again function as a template. That is, the complementary strand can also serve as a template.
  • the target is RNA
  • it can be constituted only by adding a reverse transcriptase. That is, using RNA as a template, it is possible to synthesize a complementary strand by annealing the F1 and F1c in the template by reverse transcriptase.
  • reverse transcriptase uses DNA as a template to synthesize a complementary strand
  • all reactions that synthesize a complementary strand by reverse transcriptase include the synthesis of a complementary strand that uses R1 annealed to Rlc as a starting point for synthesis. Used as a template in the displacement reaction.
  • the mode of obtaining the first single-stranded nucleic acid using RNA as a template as described above is a preferred mode of the invention.
  • a DNA polymerase having both strand displacement activity and reverse transcriptase activity such as Bca DNA polymerase
  • the same enzyme is not only synthesized from the first single-stranded nucleic acid of RNA, but is followed by DNA.
  • the reaction for the template can be similarly carried out.
  • the reaction is carried out in the presence of the following components, enabling the enzyme to react in a buffer of suitable pH, annealing or maintaining the essential salts of enzyme catalytic activity, protecting the medium of the enzyme, and regulating the melting temperature (Tm).
  • buffers for example, Tris-HCl, which has a buffering effect in the neutral or weakly alkaline range, is used.
  • the pH value is adjusted according to the DNA polymerase used, and an appropriate amount of salt, KCl, NaCl, (NH 4 ) 2 SO 4 is added to maintain the activity of the enzyme and regulate the melting temperature (Tm) of the nucleic acid, and the medium for protecting the enzyme uses bovine serum. Albumin or sugar.
  • DMSO dimethyl sulfoxide
  • Tm melting temperature
  • Modulation of the oligonucleotide is achieved by annealing of the oligonucleotide under defined temperature conditions using a melting temperature (Tm) regulator.
  • betaine N,N,N-trimethylglycine
  • tetraalkylammonium tetraalkyl
  • the desired promotion of nucleic acid amplification by the present invention can be obtained by adding 0.2-3.0 M betaine, preferably 0.5-1.5 M, to the reaction solution. Since these melting temperature regulators have the effect of lowering the melting temperature, those suitable rigor and reactivity conditions are combined with the salt concentration, reaction temperature, etc., empirically.
  • An important feature of the present invention is that a series of reactions cannot be performed unless the positional relationship of many zones is maintained. Due to this feature, non-specific synthetic reactions accompanying non-specific synthesis of complementary strands are effectively prevented. That is, even if a non-specific reaction occurs, the possibility of the product as a starting material in the subsequent amplification step is reduced, and the progress of the reaction is regulated by many regions, possibly resulting in similar nucleotides. A detection system in the sequence that accurately identifies the desired product can be constructed arbitrarily.
  • the nucleic acid synthesized by the present invention is a single strand, and in the case of a single strand, consists of a complementary nucleotide sequence, most of which are base-paired.
  • the synthesized product can be detected.
  • a fluorescent dye as a double-specific intercalater such as ethidium bromide, SYBR Green I, Pico Green or Eva Green, as the product increases, it can be observed.
  • the intensity of the fluorescence increases. By monitoring the fluorescence intensity, it is possible to track the progress of a real-time synthesis reaction in a closed system.
  • the method of synthesizing a nucleic acid of the present invention is supported by a DNA polymerase catalyzed synthesis of a strand displacement type complementary strand reaction.
  • the reaction step of the unnecessary strand displacement type polymerase is also included during the above reaction.
  • the following enzymes are known.
  • various mutants of these enzymes can be utilized in the scope of the present invention, all of which have sequence-dependent activity and strand displacement activity for complementary strand synthesis.
  • the mutant refers to a mutant including those having only the catalytic activity required to cause the enzyme or those modified by catalytic activity, stability or thermostability by, for example, mutation in an amino acid.
  • Bst DNA polymerase, Bca (exo-) DNA polymerase, and OmniAmp DNA polymerase are particularly desirable enzymes because of their degree of thermal stability and high catalytic activity.
  • the reaction of the present invention can be achieved isothermally, but due to the adjustment of the melting temperature (Tm) or the like, it is not always possible to utilize the desired temperature conditions to maintain the stability of the enzyme. Therefore, it is one of the conditions required for the thermal stability of the enzyme.
  • Tm melting temperature
  • thermal denaturation can provide nucleic acids as an initial template, and in this regard, the use of thermostable enzymes broadens the choice of protocol.
  • the various reagents necessary for the synthesis or amplification of nucleic acids of the present invention may be pre-packaged and provided in the form of a kit.
  • the kit provided by the present invention comprises a primer synthesized as a synthetic complementary strand and used for a displacement reaction.
  • Various oligonucleotides necessary for external primers, substrate dNTPs for complementary strand synthesis, DNA polymerases for strand-replacement complementary strand synthesis, buffers for providing suitable conditions for enzymatic reactions, and for detection The medium necessary to synthesize the reaction product.
  • the reagents which are added during the reaction are not required, and thus the reagents which are necessary for the reaction after the reaction to the reaction vessel, wherein the reaction can be initiated only by the addition of the sample.
  • a system for detecting a reaction product in a container by utilizing a visible light signal or a fluorescent signal It is not necessary to open and close the container after the reaction. This is very beneficial for preventing pollution.
  • the present invention synthesizes a single-stranded nucleic acid having a nucleotide sequence of a competitive stem-loop structure.
  • the nucleic acid has, for example, the following use: a first feature is the use of a specific knot having a complementary sequence in one molecule
  • a detection system that fully utilizes the characteristics of the synthetic product of the present invention can be realized by a method in which a double-strand specific intercalating agent is used in combination as a detecting reagent as described above.
  • the product of the synthesis reaction of the present invention undergoes a thermal denaturation in the detection system and returns to the original temperature, intramolecular annealing preferentially occurs, and thus allows rapid base pairing between the complementary sequences.
  • non-specific products are present, they do not have complementary sequences in the molecule such that after separation by thermal denaturation into two or more molecules, they do not immediately return to the original duplex.
  • the interference accompanying the non-specific reaction is reduced by the thermal denaturation step provided prior to the detection. If the DNA polymerase used is not resistant to heat, the thermal denaturation step has the meaning of termination of the reaction and thus facilitates control of the reaction temperature.
  • a second feature is that competitively forming hairpin loops (i.e., stem-loop structures) that are capable of base pairing are often formed.
  • the structure of a competitive hairpin loop capable of base pairing is shown in FIG. As seen in Figure 3, the loop consists of a nucleotide sequence (from the 3' to 5' end) F1c, N, R1, Nc which can be annealed intramally to form a hairpin loop.
  • a large number of base-pairable loops are supplied in a single-stranded nucleic acid.
  • a large number of probes can hybridize to one molecule of nucleic acid to allow for highly sensitive detection. It is therefore possible not only to achieve improved sensitivity but also to achieve a method for detecting nucleic acids based on specific reaction principles such as aggregation.
  • 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 particles is observed to hybridize the product to the probe.
  • the intensity of the aggregation is highly sensitive and quantitatively observed by optical measurement.
  • the aggregation can be observed by the naked eye, so that a reaction system without an optical measuring device can also be established.
  • reaction products of the invention allow for some bindable labels in which each nucleic acid molecule can be chromatographed.
  • the actual application is an analytical method (immunochromatography) using a chromatographic medium using visible detection marks.
  • the method is based on the principle that the analyte is sandwiched between an antibody immobilized on a chromatographic medium and a labeled antibody, and the unreacted labeled component is eluted.
  • the reaction product of the invention applies 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. A capture probe having a sequence complementary to a loop portion is utilized, due to the inverse of the present invention
  • the product should have a large number of hairpin loops and the product is combined with a large number of labeled probes to give a visually identifiable signal.
  • the reaction products of the present invention are often capable of providing base-paired loop regions, which can broaden various other detection systems. For example, it is feasible to use a surface cytoplasmic genome to detect a portion of the loop portion using a fixed probe. Furthermore, if the probe of the loop portion is labeled with a double-stranded specific insert, a more sensitive fluorescence assay can be performed. Or the ability to actively utilize the present invention to synthesize nucleic acids on the 3'- and 5'- sides to form base-pairing hairpins. For example, designing a loop to have a common nucleotide sequence between normal and abnormal types, and designing other loops to make a difference therein.
  • a large number of loops given by the reaction product of the present invention can be used as probes, for example, in a DNA chip, probes are densely packed in a limited area, and the technique can be fixed in a certain area.
  • the number of oligonucleotides is limited, so that a large number of annealable probes can be immobilized by high density by using the product of the present invention, that is, the reaction product of the present invention can be used as a fixed probe on a DNA chip, and the reaction product can be passed after amplification.
  • any technique known in the art can be immobilized, or a fixed oligonucleotide can be used as the oligonucleotide of the amplification reaction of the present invention, resulting in the formation of a fixed reaction product. Therefore, by using a fixed probe, a large amount of sample DNA is hybridized in a limited area, and as a result, a high signal value is expected.
  • Figure 1 is a schematic illustration of the steps of the nucleic acid synthesis method of the present invention
  • Figure 2 is a graphical representation of the steps of the second nucleic acid synthesis process of the present invention.
  • Figure 3 is a schematic illustration of the competitive hairpin structure and subsequent amplification reactions formed by the single-stranded nucleic acids of the present invention.
  • Figure 4 is a schematic illustration of the subsequent amplification reaction of nucleic acids in the present invention.
  • Figure 5 is a schematic representation of the desired amplification product formed by the synthetic method of the present invention.
  • Figure 6 is a diagram showing the positional relationship of each nucleotide sequence region corresponding to the PH5SR target nucleotide sequence in Example 1 of the present invention.
  • Figure 7 is a graph showing the real-time fluorescence curve of the PH5SR target nucleotide sequence DNA amplification process under the action of the primer of Example 1 of the present invention.
  • Figure 8 is a diagram showing the positional relationship of each nucleotide sequence region corresponding to the target nucleotide sequence of the MERS-orf1a in Example 2 of the present invention.
  • Figure 9 is a photograph showing the results of agarose electrophoresis of the product obtained by the method for synthesizing the single-stranded nucleic acid of the present invention using MERS-orf1a as a template in Example 2 of the present invention; wherein, Lane 1: Biyuntian O0107 DNA Ladder; Lane 2: 1fmol MERS- Orf1a dsDNA.
  • Figure 10 is a photograph showing the results of agarose gel electrophoresis of a restriction enzyme digestion product in Example 3 of the present invention, wherein the product was obtained in Example 2 by the nucleic acid synthesis reaction of the present invention. among them,
  • Lane 5 molecular weight marker DNA ladder.
  • Figure 11 is a graph showing the real-time fluorescence of the MERS-orf1a target nucleotide sequence DNA amplification process under the action of the primers in Example 4 of the present invention.
  • Figure 12 is a graph showing the real-time fluorescence curve during RNA amplification of the MERS-orf1a target nucleotide sequence in vitro under the action of the primers in Example 5 of the present invention.
  • Fig. 13 is a view showing the action site of the amplification principle of the target nucleotide in the sixth embodiment of the present invention with the addition of the acceleration probe.
  • Figure 14 is a graph showing the fluorescence intensity of the MERS-orf1a system as a function of reaction time under the action of different combinations of accelerated probe primer combinations in Example 6 of the present invention.
  • Figure 15 is a graph showing the real-time fluorescence curve during DNA amplification of the target nucleotide sequence of the influenza A virus H1 gene under the action of the designed influenza A virus H1 target nucleotide primer in Example 7 of the present invention.
  • Fig. 16 is a graph showing the real-time fluorescence curve during DNA amplification of the genus Herpes simplex virus type III target nucleotide sequence under the action of the genus Herpes simplex virus type III target nucleotide primer in Example 8 of the present invention.
  • the smallest target fragment may be a nucleic acid sequence of only 60 bases.
  • the 5S rRNA gene Genbank: HQ681111
  • the crystal pudding fish Pieris spongid fish
  • Food Chem. 2011; 129: 1860-4. the target gene, and achieved the application of the method of the present invention. Constant temperature amplification of the target gene.
  • the nucleic acid of the present invention having a complementary strand joined to the single strand in the form of a helical loop was attempted using a man-made design of Pangasianodon hypophthalmus 5S rRNA (abbreviated as PH5SR) (from GenBank: HQ681111) with an enzyme cleavage site.
  • PH5SRNF nucleotide sequence as shown in SEQ ID NO. 1
  • PH5SRNR nucleotide sequence as shown in SEQ ID NO. 2
  • each region of the target nucleotide sequence is shown in Figure 2.
  • primers PH5SRNF and PH5SRNR Two Nc segments were synthesized at both ends of the target nucleotide PH5SR to compete with the N segment on the target nucleotide sequence to form a hairpin loop, and the synthesis process is shown in FIG. 2 .
  • the combination of the reaction solutions of the method of synthesizing the nucleic acid of the present invention by these primers is shown below.
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • Target nucleic acid PH5SR dsDNA (nucleotide sequence is shown in SEQ ID NO. 3). Referring to Figure 6, the positional relationship of each nucleotide sequence region corresponding to the nucleotide sequence of the PH5SR target.
  • the nucleic acid of the present invention having a complementary strand joined to the single strand in the form of a helical loop was attempted by using an artificially designed MERS-orf1a (from GenBank: KX108946.1) inserted with an enzyme cleavage site as a template.
  • Two primers used in the experiment were Mo1aNF (nucleotide sequence as shown in SEQ ID NO. 4) and Mo1aNR (nucleotide sequence as shown in SEQ ID NO. 5). These are designed to anneal to a zone of the ring by utilizing adjacent stacking phenomena. Furthermore, setting these primers to a high concentration preferentially causes annealing of Mo1aNF (or Mo1aNR).
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • Target nucleic acid MERS-orf1a dsDNA (nucleotide sequence is shown in SEQ ID NO. 6). Referring to Figure 8, is the positional relationship of each nucleotide sequence region corresponding to the target nucleotide sequence of MERS-orf1a.
  • the mixture was reacted at 63 ° C for 1 hour, and after the reaction, the reaction was terminated at 80 ° C for 10 minutes, and then transferred to water pre-cooled with ice.
  • nucleic acid product having a broad molecular weight distribution was obtained, i.e., the nucleic acid obtained by the inventive method was verified to be infinitely self-assembled and annealed to obtain an oversized nucleic acid molecule.
  • Example 3 confirms the reaction product by restriction enzyme digestion
  • Example 2 of the present invention having a complementary nucleotide sequence linked in a single chain in a cyclic structure
  • the product was digested with a restriction enzyme. If the theoretical fragment is generated by digestion, and at the same time, the high molecular weight observed in Example 2 produces an unclear strip pattern and a band that is not electrophoresed, any of these products can be expected to be present.
  • a nucleic acid having a complementary sequence alternately linked within a single strand is invented.
  • Example 2 The reaction solution in Example 2 was deposited and purified by phenol treatment and ethanol precipitation, and the resulting precipitate was recovered and redissolved in ultrapure water, and the restriction enzymes HindIII, XhoI and the two enzymes were used for digestion at 37 ° C. Hours, samples at 90mV pre-stained in GelRed (Biotum) 1% agarose gel (TAE dissolution) was electrophoresed for 1 hour.
  • the Biyuntian O0107 DNA Ladder was used as the molecular weight marker. The gel after electrophoresis is used to verify the nucleic acid.
  • EvaGreen is a dye with a green excitation wavelength that binds to all dsDNA double helix minor groove regions, and its inhibition of nucleic acid amplification reactions such as PCR is much smaller than the latter.
  • EvaGreen emits a weak fluorescence, but once bound to double-stranded DNA, the fluorescence is greatly enhanced. Therefore, the fluorescence signal intensity of EvaGreen is related to the amount of double-stranded DNA, and the amount of double-stranded DNA present in the nucleic acid amplification system can be detected based on the fluorescence signal.
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • Target nucleic acid MERS-orf1a dsDNA (nucleotide sequence is shown in SEQ ID NO. 6).
  • Set ABI StepOne real time PCR reaction temperature is constant at 63 ° C, reaction time is 60 min.
  • the curve of the fluorescence intensity as a function of reaction time is shown in FIG. Fluorescence detection is applied to the purpose of real-time monitoring, and the results can be judged in advance by real-time amplification curves.
  • AMV reverse transcriptase can synthesize cDNA using RNA as a template, and Bst DNA polymerase can detect RNA.
  • the cDNA was synthesized by using the primer Mo1aNF (nucleotide sequence shown in SEQ ID NO. 4) and Mo1aNR (nucleotide sequence shown in SEQ ID NO. 5) using RNA as a template, and the reaction solution was combined as follows, and the rest was ddH 2 O. Up to 25 ⁇ L
  • MERS-orf1a RNA RNA nucleic acid sequence is shown as SEQ ID NO. 7
  • the MERS-orf1a RNA is obtained by in vitro transcription of MERS-orf1a (sequence as shown in SEQ ID NO. 6).
  • the accelerated probe combination is divided into four groups, only the primer combinations are different (where the accelerated probe 1 refers to the primers F2 and R2, and the accelerated probe 2 refers to the primers Fin and Rin):
  • FIG. 13 there is shown a schematic diagram of the action site for the amplification of target nucleotides with the addition of an accelerated probe.
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • the target nucleic acids corresponding to the primers of each group a, b, c, and d are: MERS-orf1a dsDNA (the sequence is shown in SEQ ID NO. 6).
  • Set ABI StepOne real time PCR reaction temperature is constant at 63 ° C, reaction time is 60 min.
  • the curve of the fluorescence intensity as a function of reaction time is shown in FIG.
  • the H1N1 virus belongs to the Orthomyxoviridae family, Influenza virus A.
  • the symptoms of influenza A H1N1 are similar to those of a cold. Patients may have fever, cough, fatigue, and loss of appetite.
  • H1N1 was popular in a large area, causing a certain degree of panic. Because of the nucleic acid detection of H1N1, cDNA is generally detected by reverse transcription and PCR. Therefore, the application of the method of the present invention to design new primers can also be applied to the detection of H1N1 virus.
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • the target nucleic acid is: H1dsDNA (sequence is shown in SEQ ID NO. 14)
  • Example 8 for the amplification of the target gene of the herpes simplex virus III
  • the koi herpes virus disease, herpes virus hematopoietic organ necrosis, and acne sore disease caused by herpes virus infection in the carp family are serious threats to carp culture.
  • the virus is highly pathogenic and highly contagious, causing the disease to be prevalent worldwide, and the mortality rate of infected fish can reach 80% to 100%.
  • the disease has attracted the attention of the International Organization for Animal Health (OIE) and listed it as a list of key epidemics.
  • OIE International Organization for Animal Health
  • China has classified the disease as a second-class animal disease and has carried out daily monitoring work. It is very important to develop corresponding testing techniques to achieve rapid detection of relevant epidemics and to respond to epidemics. Therefore, the method of the present invention is selected as a potential application target.
  • reaction solution is combined as follows, and the rest is ddH 2 O to 25 ⁇ L.
  • the target nucleic acid is: CyHVIII dsDNA (sequence as shown in SEQ ID NO. 17)

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Abstract

提供了一种恒温条件下合成核酸的方法及试剂盒，所述方法为：1)提供一种核酸，所述核酸的5'末端具有可与同一条链上的N区退火的Nc区，同时该核酸的3'末端具有可与同一条链上的N区退火的Nc区，此核酸的5'末端和3'末端所具有的Nc区与同一链上的N区退火存在竞争关系；2)以步骤1)所述核酸为模板，将已与N区退火的Nc区的3'端为合成起点，合成其自身的互补链；3)通过聚合酶催化链置换型互补链合成反应进行互补链合成，以置换步骤2)中所合成的互补链。本发明的核酸合成方法可针对短链核酸片段(理想片段可仅为60bp)应用单酶的恒温体系实现基因的快速扩增。

Description

一种恒温条件下合成核酸的方法及试剂盒 技术领域
本发明涉及基因工程技术领域，具体地，本发明涉及一种恒温条件下合成核酸的方法，特别涉及由特异性核苷酸序列构成的可形成特殊结构核酸的合成方法，及基于该特异性核酸序列的一种有用的扩增核酸的方法。
背景技术
生物体所携带基因信息的生物体的最根本差异，基于核苷酸序列互补性的分析方法可直接分析基因所携带的遗传特征。该分析是一种鉴定遗传疾病、癌变、微生物等非常强有力的方法。当样品中靶基因含量非常少时一般不易检测，因此必须对靶基因进行扩增或使其检测信号放大。作为扩增靶基因的方法，PCR方法被认为是最经典方法(Saiki，Gelfand et al.1988)，亦是体外核酸序列扩增应用最为普遍的技术。该方法指数式扩增结果使其具有高灵敏度，确立了其在分子生物学方法检测领域的地位，经几十年发展已有系列成熟产品。此外，扩增产物可回收使用，因此作为一种支持遗传工程技术如基因克隆和结构决定的重要工具，亦得到广泛应用。然而，PCR方法明显有下述的问题：实际操作中必须要用专门的程序温度控制系统；扩增反应的指数式上升导致难于定量；样品和反应溶液易受到外部污染，假阳性问题较为突出。
例如：一旦PCR中偶然误合成互补链，在接下去的反应中该产物以模板运行，就会造成错误的结果。实际应用中，若引物末端仅一个碱基不同则将很难严格控制PCR，故必须改进特异性使PCR更好应用于SNPs的检测上。另一方面，相较于繁琐的程序温控过程合成核酸，科学家亦开发出在恒温条件下合成核酸的技术(Zhao，Chen et al.2015)，主要包括以下几种：依赖核酸序列的扩增(NASBA)、滚环扩增(RCA)，链置换扩增(SDA)、环介导等温扩增(LAMP)、依赖解旋酶的扩增(HDA)、重组聚合酶扩增(RPA)。
NASBA，也称TMA(转录介导扩增方法)不需要复杂的温度控制。该方法通过DNA聚合酶以靶RNA为模板，加入连接有T7启动子的探针而得以合成，第二探针进入双链使产物得以生成，接着以生成的双链DNA为模板通过T7RNA聚合酶转录而扩增得到大量的RNA产物。NASBA需要热变性步骤直到双链DNA形成，但是接下去的转录反应在等温条件下通过T7RNA聚合酶得以进行。必须要用多种酶组合例如反转录酶，RNase H，DNA聚合酶和T7RNA聚合酶，然而多种酶的组合对于费用是不利的。同时由于复杂的多种酶反应条件设定，该方法很难作为一般的分析方法得以推广。
RCA(滚环扩增，Rolling Circle Amplification)旨在模仿微生物中环状DNA的滚环复制过程，对于环状单链DNA模板，与该模板相结合的引物在原方法仅能实现对于环状核酸的扩增。为使得该方法可通用于线性DNA的扩增，通过挂锁探针(padlock probe)或环形探针互补的单链DNA显示具有一系列核苷酸序列与挂锁探针(padlock probe)或环形探针互补的单链DNA在靶核苷酸存在下可被连续的合成(Lizardi，Huang et al.1998)。该方法亦存在需多种酶的问题。而且，互补链合成的启动取决于连接两邻近区的反应，并且其特异性基本上与LCR中的相同。
链替代扩增(Strand Displacement Amplification，SDA)方法也是人们所知的扩增具有序列与靶序列互补的模板DNA的方法(Zhang，Cui et al.1992)。SDA方法应用特定的DNA聚合酶从目标核苷酸序列3’-侧互补的引物开始合成互补链以替换双链5’-侧的序列。由于新合成的互补链替换了5’-侧的双链，称该项技术为SDA方法。SDA方法中限制酶识别序列作为引物预先插入到退火序列中就可去除PCR方法中必须的温度变化步骤。即通过限制酶生成的切口供给3’-OH基作为互补链的合成起点，并且先合成的互补链通过链置换合成得以释放单链，接着再次作为模板用于下面的互补链合成。但SDA扩增产物与天然核酸结构不同，并且对用限制酶来断裂或将扩增产物应用到基因克隆上存在限制。这方面也是导致费用较高的主要原因。另外，SDA方法应用在未知序列时，用于引入缺口的限制酶识别序列相同的核苷酸序列可能存在于要被合成的区中，如此可能阻止完全互补链的合成。
依赖解旋酶DNA恒温扩增技术(Helicase-dependent Isothermal DNA Amplification，HDA)是由美国NEB公司研究人员于2004年发明的一种新型核酸恒温扩增技术(Vincent，Xu et al.2004)。该技术模拟自然界生物体内DNA复制的自然过程，在恒温条件下利用解旋酶解开DNA双链，同时DNA单链结合蛋白(single-stranded DNA-binding protein，SSB)用以稳定解开的单链，并为引物提供结合模板，然后由DNA聚合酶催化合成互补链。新合成的双链在解旋酶的作用下又解成单链，并作为下一轮合成的模板进入上述的循环扩增反应，最终实现靶序列的指数式增长。
重组酶聚合酶扩增(Recombinase Polymerase Amplification，RPA)，其要点在于：重组酶与引物结合形成的蛋白-DNA复合物，能在双链DNA中寻找同源序列。一旦引物定位了同源序列，随后链置换DNA聚合酶即会介导链交换反应形成并启动DNA合成，对模板上的目标区域进行指数式扩增。被替换的DNA链与单链结合蛋白(SSB)结合，以防止进一步替换。在这个体系中，由两个相对的引物起始一个合成事件。整个过程进行得非常快，一般可在十分钟之内获得可检出水平的扩增产物。但整个过程中需要筛选能与重组酶的结合并且特异性良好的引物，同时需要使用三种酶将极大增加其成本，设计引物的难度也较大。
LAMP技术(Notomi，Okayama et al.2000)的核心是利用针对靶基因上的六个区域设计四条特异性引物依靠一种高活性链置换DNA聚合酶，使得链置换DNA合成在不停地自我循环。该技术可在15-60分钟内实现109-1010倍的扩增，反应能产生大量的扩增产物即焦磷酸镁白色沉淀，可以通过肉眼观察白色沉淀的有无来判断靶基因是否存在，日本荣研公司亦针对性开发出浊度仪以实现扩增反应实时监测。LAMP方法的优势除了高特异性和高灵敏度外，操作还十分的简单，在应用阶段对仪器的要求低，一个简单的恒温装置就能实现反应，结果检测也非常简单，直接肉眼观察白色沉淀或者绿色荧光即可，是一种适合现场、基层快速检测的方法。其中一个局限是，因该方法高特异性和灵敏度等特点依赖于4条引物的性质，最佳引物的获得通常需要进行序列比对、在线引物设计、引物筛选及特异性试验，这一过程十分繁 琐。同时LAMP技术所需的靶基因片段亦较大，对于短链核酸难于适用。例如，5S rRNA是由120个左右的核苷酸单位所构成，在所有的生命体中均为重要的结构性和功能性核糖体亚基。5S rRNA具有高度的保守性，故常被选择为分子检测的标志物。由于5S rRNA的核酸序列较短，现在针对此标志物的检测一般采用PCR的方法。而核酸等温扩增具有简单快捷的优势，但难于应用于5S rRNA的检测。以环介导恒温扩增(LAMP)为例，LAMP引物为F3，B3，FIP和BIP构成，设计这些引物所需的核酸序列就需要120左右的碱基，更不用说设计F2到B2之间的间隔了，难于满足LAMP引物设计的苛刻条件，故不能应用于5S rRNA的检测。
发明内容
本发明的目的是提供一种合成核酸的方法，受到DNA常见双螺旋结构、分子信标探针及LAMP启发的新原理，以PCR引物为基础重新设计并规划了引物工作模式，形成竞争性茎环起始扩增结构，设计该结构环的形成过程，并且具有可选使用外引物的特点。更具体的是提供一种新型的依靠序列高效合成核酸的低成本方法。亦即，本发明的目的是提供通过在一种单酶及等温条件下完成核酸合成和扩增的方法。本发明的另一目的是提供一种核酸合成的方法，该方法通过改造已有的核酸合成反应原理很难达到的高特异性的核酸快速合成，还提供一种用该合成方法扩增核酸方法。本发明的一个优势是可针对短链核酸片段(理想片段可仅为60bp，较LAMP最小的理想片段120bp更短)应用单酶的恒温体系实现基因的快速扩增。本发明利用聚合酶催化链置换型的互补链合成而不需复杂的温度控制，有益于核酸的合成。该DNA聚合酶是SDA、RCA、LAMP等方法中用到的酶。
本发明人改进了已知方法3’-OH的供给，结果发现通过利用具有特定结构的寡核苷酸，不需任何额外酶反应3’-OH结构就可被提供，由此得出本发明。即本发明涉及合成核酸的方法，通过用所述核酸合成方法扩增核酸的方法和应用所述方法合成核酸的试剂盒。
本发明的具体技术方案如下：
本发明提供一种恒温条件下合成核酸的方法，包括以下步骤：
1)提供一种核酸，所述核酸的5’末端具有可与同一条链上的N区退火的Nc区，同时该核酸的3’末端具有可与同一条链上的N区退火的Nc区，此核酸的5’末端和3’末端所具有的Nc区与同一链上的N区退火存在竞争关系；
2)以步骤1)所述核酸为模板，将已与N区退火的Nc区的3’端为合成起点，合成其自身的互补链；
3)通过聚合酶催化链置换型互补链合成反应进行互补链合成，以置换步骤2)中所合成的互补链。
所述恒温条件下合成核酸的方法，具体合成步骤如下：
1)提供一种核酸的步骤，该核酸的5’末端和3’末端均具有能够与同一条链上的N区退火的Nc区；所述3’末端的Nc区与N区退火时能形成环，所述环包含能进行碱基配对的F1c区；所述5’末端的Nc区与N区退火时能形成环，所述环包含能进行碱基配对的R1区；所述核酸的5’末端和3’末端所具有的Nc区与同一链上的N区的退火存在竞争关系；
2)使第一寡核苷酸I与步骤1)提供的所述核酸的F1c区退火，然后以所述第一寡核苷酸I的F1区作为合成起点，进行合成步骤；其中所述第一寡核苷酸I包括N区与Fl区；
3)以步骤1)提供的所述核酸为模板合成其自身的互补链，将已与N区退火的Nc区的3’端为合成起点；将合成完后的核酸序列称为核酸A；
4)使第二寡核苷酸II与步骤3)提供的所述核酸A的R1c区退火，然后以所述第二寡核苷酸II的R1区作为合成起点，进行合成步骤；其中所述第二寡核苷酸II包括R1区和Nc区；
5)以步骤3)提供的所述核酸A为模板合成其自身的互补链，将3’端已与Nc区退火的N区为合成起点，获得在其一条链上具有首尾互补核苷酸序列的核酸，并且该核酸链上具有交替连接的互补核苷酸序列区。
参见图1，为本发明中上述合成核酸所对应的步骤图解。
优选地，上述步骤1)所述核酸的制备方法，包括以下步骤：
1-1)退火步骤，使第一寡核苷酸I与模板的F1c区退火，其中该模板的3’末端包括F1c区和位于F1c区5’侧的N区，该模板的5’末端包括R1区，其中所述第一寡核苷酸I包括N区与Fl区，所述N区与F1区的5’侧相连，其 中，
F1区：具有与F1c区互补的核苷酸序列的区，
N区：与Nc区互补的核苷酸序列的区；
1-2)以所述第一寡核苷酸I的F1区作为合成起点，合成第一核酸；所述第一核酸具有与所述模板互补的核苷酸序列，所述第一核酸的5’末端具有可与同一条链上的Nc区退火的N区，并且通过所述Nc区与N区的退火可形成茎环；
1-3)利用聚合酶催化链置换反应置换获得步骤1-2)所合成的第一核酸；
1-4)退火步骤，使第二寡核苷酸II与步骤1-3)所得第一核酸的R1c区退火，其中所述第二寡核苷酸II包括R1区和Nc区，并且Nc区与R1区的5’侧相连；其中，
R1区：具有与R1c区互补的核苷酸序列的区，
Nc区：与N区互补的核苷酸序列的区；
1-5)以所述第二寡核苷酸II的R1区作为合成的起点，合成第二核酸；
1-6)利用聚合酶催化链置换反应置换所述第二核酸获得步骤1)所述的核酸。
参见图2，为本发明中上述第二核酸(即步骤1)中所述的核酸)所对应的步骤图解。
优选地，所述步骤1-1)所述模板为RNA，步骤1-2)中的第一核酸通过具有反转录酶活性的酶来合成。
本发明适用于各种DNA及RNA，例如各种动植物细胞、细菌和病毒的DNA及RNA等等。比如，用于H1N1病毒的H1基因和N1基因的cDNA及RNA检测；用于MERS-CoV病毒的cDNA及RNA检测，如：该RNA的orf1a，orf1b区段；用于鲤疱疹病毒III型的DNA检测等。
优选地，所述F1c区、N区和R1区的核酸片段均为15-60bp。进一步优选地，所述F1c区、N区和R1区的核酸片段均为20bp。
所述恒温条件下合成核酸的方法，还包括：所述步骤5)获得的核酸链能够自主配对无限延伸，该核酸链上的3’端Nc区会与该链上的互补区段N区配对作为合成起点以自身为模板使所述核酸链不断延伸。
根据本发明所述的合成核酸的方法，其中，所述合成的核酸是指在 其一条链上具有首尾互补核苷酸序列的核酸。
根据本发明所述的合成核酸的方法，其中，所述恒温是指整个反应过程在60-65℃的温度范围内进行合成。
根据本发明所述的合成核酸的方法，作为优选，通过引入加速引物X2和/或Xin的方法使得核酸扩增加速进行；其中X2是位于原始核酸互补链的F1区及R1区的5’侧的区段，Xin是位于F1c区到N区及Nc区到R1c的中间区段。本发明所述聚合酶催化链置换反应中使用的聚合酶为Bst DNA聚合酶、Bca(exo-)DNA聚合酶、DNA聚合酶I克列诺(Klenow)片段、Vent DNA聚合酶、Vent(Exo-)DNA聚合酶(缺少核酸外切酶活性的Vent DNA聚合酶)、Deep Vent DNA聚合酶、Deep Vent(Exo-)DNA聚合酶(缺少核酸外切酶活性的Deep Vent DNA聚合酶)、Φ29phage DNA聚合酶以及MS-2phage DNA聚合酶等中的一种或几种。其中优选使用Bst DNA聚合酶或Bca(exo-)DNA聚合酶。
根据本发明所述的合成核酸的方法，其中，所述聚合酶催化链置换反应中可以加入解链温度调节剂。其中，所述解链温度调节剂优选为甜菜碱，进一步优选地，反应溶液中甜菜碱的浓度为0.2-3.0M。
本发明的用于恒温条件下合成核酸的试剂盒，其特征在于，所述试剂盒包括：
第一寡核苷酸I，其包括F1区和N区，所述N区与F1区的5’侧相连，其中，
F1区：具有与F1c区互补的核苷酸序列的区，和
N区：与Nc区互补的核苷酸序列的区；
第二寡核苷酸II，其包括R1区和Nc区，所述Nc区与R1区的5’侧相连，其中，
R1区：具有与R1c区互补的核苷酸序列的区，和
Nc区：与N区互补的核苷酸序列的区；
催化链置换型互补链合成反应的DNA聚合酶，和，
核苷酸，其作为所述DNA聚合酶的底物。
根据本发明所述的试剂盒，其中，所述试剂盒还包含用于检测核酸 合成反应产物的检测试剂。
根据本发明所述的试剂盒，其中，所述试剂盒还包括加速引物X2和/或Xin，其中X2是位于原始核酸互补链的F1区及R1区的5’侧的区段，Xin是位于F1c区到N区及Nc区到R1c的中间区段。
根据本发明所述的试剂盒，其中，所述DNA聚合酶为Bst DNA聚合酶、Bca(exo-)DNA聚合酶、DNA聚合酶I克列诺片段、Vent DNA聚合酶、Vent(Exo-)DNA聚合酶、Deep Vent DNA聚合酶、Deep Vent(Exo-)DNA聚合酶、Φ29phage DNA聚合酶以及MS-2phage DNA聚合酶等中的一种或几种。其中优选使用Bst DNA聚合酶或Bca(exo-)DNA聚合酶。
本发明提供了上述试剂盒在合成核酸或检测样品中靶核苷酸序列中的应用。
基于本发明的合成核酸的方法，提供一种检测样品中靶核苷酸序列的方法，包括以靶核苷酸为模板，通过本发明的合成核酸的方法进行扩增，并观察是否生成扩增产物。
在上述扩增产物中加入包含与形成的茎环结构互补的核苷酸序列的探针，进而观察两者之间的杂交。还可以将所述探针标记在颗粒上，并观察通过杂交而发生的聚集反应。所述的扩增方法可以在核酸检测试剂的存在下实施，并根据该检测试剂的信号变化来观察是否生成扩增产物。
基于本发明的合成核酸的方法，还可以提供一种检测样品中靶核苷酸序列突变的方法，包括以靶核苷酸为模板，通过本发明所述的合成核酸的方法进行扩增。其中，核苷酸序列中作为扩增对象的突变阻碍了组成该扩增方法的任一互补链的合成，从而检测出突变。
可形成竞争性茎环结构的核苷酸序列的单链核酸是本发明合成的目的，该核酸指的以靶核酸序列为具有互相互补核苷酸序列以一定次序交替连接在单链里的核酸。本发明合成的核酸基本上由通过成茎环结构连接的互相互补的链组成。参见图5，为本发明合成方法形成的理想扩增核酸产物的示意图。
一般而言，不管是否部分涉及碱基配对，一个在配对碱基分离时不 能被分离成两个或更多分子的链称为单链。同一链中互补核苷酸序列可形成碱基配对，本发明通过容许具有核苷酸序列首尾连接在单链里的核酸在同一链内碱基配对，可获得分子内碱基配对的产物，该产物包含组成明显双链的区和不涉及碱基配对的环。
也就是，本发明具有竞争性茎环结构的核苷酸序列的核酸可被定义为单链核酸，其中包含能在同一链中退火的互补核苷酸序列。具有互补核苷酸序列的核苷酸可退火成不涉及碱基配对的环。成环序列可以是任意的核苷酸序列。成环序列能碱基配对以启动用于置换的互补链的合成。并优先地被提供与位于其它区的核苷酸序列不同的序列，以获得特异性退火。
本发明中基本相同的核苷酸序列定义如下：当以某序列作为模板合成的互补链与靶核苷酸序列退火以供给合成互补链的起点时，该某序列基本上与靶核苷酸序列相同。例如，基本上与F1相同的序列不但完全包括与F1相同的序列，还包括能作为模板的核苷酸序列，所述模板能给出与F1退火的核苷酸序列并能作为合成互补链的起点。本发明术语“退火”指的是通过根据沃森-克里克定律的碱基配对，形成互补结构的核酸。故而，即使组成碱基配对的核酸链为单链，如果分子内互补核苷酸序列碱基配对，退火亦会发生。通过碱基配对核酸组成双链结构，故本发明退火和杂交所表达的含义有重合部分。
本发明组成核酸的核苷酸序列对数至少为1。本发明所期望的模型中，核苷酸序列对数可为1的整倍数。该情况中，本发明组成核苷酸的互补核苷酸序列对数理论上没有上限，在由多组互补核苷酸序列构成的本发明合成产物核酸时，该核酸由重复相同的核苷酸序列组成。
本发明合成的具有竞争性茎环结构的核苷酸序列的单链核苷酸与天然存在的核酸具有不同的结构，并且一般已知当通过核酸聚合酶作用合成核酸时如果用核苷酸衍生物作为底物，就可合成核酸衍生物。所用核苷酸衍生物包括放射性同位素标记的核苷酸或结合配体标记的核苷酸衍生物例如生物素或地高辛。这些核苷酸衍生物可用于标记产物核酸。或 者，如果底物是荧光核苷酸，则产物核酸可能为荧光衍生物。
利用DNA聚合酶能启动有上述结构的核酸的合成，该DNA聚合酶具有链置换活性以及Nc区具备在3’-末端与同一链上的部分N区退火进而合成互补链。有许多关于互补链合成反应的报道，其中形成螺旋环，以螺旋环序列自身为模板，亦有形成发夹环，以发夹环序列自身为模板，而本发明中提供给竞争性发卡环其部分能碱基配对的区，并且具有在合成互补链时利用该区的新特点。通过将该区用作合成的起点，先前以发卡环序列自身为模板合成的互补链被替换。
本发明用到术语“核酸”，本发明核酸通常既包括DNA又包括RNA。然而，本发明用于合成核酸的模板，来自天然DNA或RNA的其核苷酸被人工衍生物所替换的核酸或修饰核苷酸亦包括在本发明的核酸范围中。通常本发明的核酸被包含于生物样品中，生物样品包括动物，植物或微生物的组织，细胞，培养物和分泌物，以及它们的提取物。本发明的生物样品包括细胞内寄生物基因组DNA或RNA，例如病毒或支原体。本发明的核酸一般由包含在所述生物样品的核酸衍生而来。例如由mRNA合成cDNA，基于生物样品衍生来的核酸而扩增的核酸，是本发明的核酸的典型实例。
本发明核酸的特征是在3’-及5’-末端被提供Nc区，可与同一链上的部分N区退火，通过该Nc区与同一链上的N区退火，可形成发卡环，可在各种方法中得到该核酸，所述核酸的5’末端和3’末端所具有的Nc区与同一链上的N区的退火存在竞争关系。此外仅在3’-末端的Nc区同N区退火时才可在DNA聚合酶的作用下延伸。参见图3，为本发明单链核酸所形成的竞争性发卡结构及后续扩增的示意图。图4为核酸后续扩增反应的示意图。其中，图3和图4中的NF即是指第一寡核苷酸I，图4中的NR即是指第二寡核苷酸II。
组成基于本发明所述寡核苷酸的核苷酸序列特征所用术语“相同的”和“互补的”并不意味着绝对相同和绝对互补。也就是，与某序列相同的序列包括与某序列退火的核苷酸序列互补的序列。另一方面，互补序 列是严格条件下能退火的序列，提供作为互补链合成的起点3’-末端。
本发明中，寡核苷酸是满足两个要求的核苷酸，即必须能形成互补碱基配对，并且在3’-末端供给-OH基为互补链合成的起点。因此，其主链并不必限于磷酸二酯键一种连接。例如，它可由硫代磷酸衍生物组成主链或者是基于肽连接的肽核酸，所述硫代磷酸衍生物为S取代O。碱基是指那些可互补配对的碱基。天然存在五种碱基，即A，C，T，G和U，碱基也可为类似物例如溴脱氧尿苷。优选的是，本发明寡核苷酸不仅可用做合成的起点还可为互补链合成的模板。本发明术语多核苷酸包括寡核苷酸。本发明所用术语“多核苷酸”的链长无限制，而所用术语“寡核苷酸”指的是有相对较短的链长的核苷酸聚合物。
下述各种核酸合成反应中在给定的条件下，本发明寡核苷酸链有能与互补链碱基配对并保持一定的特异性这样一种长度。具体地，它由5-200个碱基组成，更优选10-50个碱基对。识别已知聚合酶的链长至少为5个碱基。该聚合酶催化依靠序列的核酸合成反应。故而退火部分的链长应长于该长度。另外，统计学上所期望10个碱基的长度或更长以获得目标核苷酸特异性。另一方面，由于化学合成制备太长核苷酸序列比较困难。因此上述链长是所期望范围的实例。例证的链长指的是部分与互补链退火的链长。正如下面所描述的，本发明寡核苷酸可最终至少分别与两区退火。因此，这里例证的链长应理解为组成寡核苷酸的每个区的链长。
此外，本发明的寡核苷酸可用已知的标记物标记。标记物包括结合配体例如地高辛和生物素，酶，荧光物，发光物，放射性同位素。众所周知通过荧光类似物替换组成寡核苷酸的碱基的技术(W095/05391，Proc.Natl.Acad.Sci.USA，91，6644-6648，1994)。
本发明其它寡核苷酸还可被结合到固相。或者，寡核苷酸的任意部分可用结合配体标记，例如生物素，间接地由结合配体例如固定抗生物素蛋白所固定。固定寡核苷酸为合成的起点时，合成反应产物核酸为固相所捕获，这将利于其分离。通过核酸特异性指示物或与标记探针的杂 交可对分离部分进行检测。通过本方法所获得的核酸产物，针对其中靶核酸片段可通过限制酶消化产物而得以回收。
本发明所用术语“模板”是指用于合成互补链时作为模板的核酸。具有核苷酸序列与模板互补的互补链意思是指对应于模板的链。但是二者的关系只是相对的。即合成的互补链可以再次起到模板的功能。也就是，互补链亦可作为模板。
在本发明中，如果目标为RNA，可仅通过添加反转录酶构成。即用RNA为模板，通过反转录酶在模板中F1与F1c的退火有可能合成互补链。当反转录酶以DNA为模板进行合成互补链的反应时，所有通过反转录酶进行合成互补链的反应包括以与Rlc退火的R1作为合成起点的互补链的合成，该互补链在链置换反应中作为模板。如上述以RNA为模板获得第一条单链核酸的模式为本发明的优选模式。另一方面，如果使用既具有链置换活性又有反转录酶活性的DNA聚合酶如Bca DNA聚合酶，通过相同的酶不但从RNA的第一条单链核酸的合成，而且接下去以DNA为模板的反应可得以类似地进行。
反应在下面成分存在下进行，能使酶反应处于合适pH值的缓冲液，退火或维持酶催化活性的必需盐，保护酶的介质以及调控解链温度(Tm)所必须的调控物。对于缓冲液，例如所用的在中性或弱碱性范围有缓冲作用的Tris-HCl。根据所用的DNA聚合酶调节pH值，对于盐，KCl，NaCl，(NH4)2SO4等适量加入以保持酶的活性并调控核酸的解链温度(Tm)，保护酶的介质使用牛血清白蛋白或糖类。此外，一般用二甲基亚砜(DMSO)或甲酰胺作为解链温度(Tm)的调控物。通过利用解链温度(Tm)的调控物在限定的温度条件下寡核苷酸的退火得到了调控。而且，甜菜碱(N，N，N-三甲基甘氨酸)或四烷基铵盐(tetraalkyl)通过其等稳定作用(isostabilization)对于改善链置换的效率也是有效的。通过向反应溶液中加入0.2-3.0M甜菜碱，优选0.5-1.5M，可得到所希望的本发明对核酸扩增的促进作用。因为这些解链温度的调控物有降低解链温度的作用，那些合适的严谨性和反应性条件要结合盐的浓度，反应 温度等凭经验而定。
本发明重要的特征是除非许多区的位置关系得以保持，否则一系列反应不能进行。由于这个特征，伴随互补链非特异合成的非特异合成反应得到了有效阻止。也就是即使发生某非特异反应，在合成接下去的扩增步骤中产物作为起始物质的可能性也得到了降低，而且，通过许多区调控反应的进展，有可能导致在类似的核苷酸序列中能精确的鉴定出所需产物的检测系统可被随意地组成。
本发明合成的核酸是单链，就单链而言，由互补核苷酸序列构成，其大部分均为碱基配对的。通过利用这个特征，对合成的产物可进行检测。通过实施本发明合成核酸的方法，在有荧光色素作为双链特异性嵌入剂(double-specific intercalater)例如溴化乙锭、SYBR Green I、Pico Green或Eva Green，随着产物的增加可观察到荧光的强度增加。通过监测荧光强度，就可能在封闭系统中跟踪实时(real-time)合成反应进行情况。也可考虑在PCR方法中应用该类型的检测系统，但有许多问题，因为不能区分产物信号和引物二聚物的信号等。然而，当本发明应用该系统时，增加非特异碱基配对的能力非常低，因此，预计高灵敏度和低干扰可能同时能获得，与应用双链特异性嵌入剂(double-specific intercalater)相似，在同一系统中可利用荧光能量的转移用于实现检测系统的方法。
本发明合成核酸的方法通过DNA聚合酶催化合成链置换型的互补链反应而得到支持。上述反应期间，也包含不必需链置换型聚合酶的反应步骤。然而，为了组成试剂的简单化及经济的观点，使用一种DNA聚合酶有利，该种DNA聚合酶，下列的酶是已知的。此外，本发明范围中可利用这些酶的各种突变体，它们都具有用于互补链合成的序列-依赖活性和链置换活性。其中突变体指的是包括那些仅具有导致酶所需的催化活性的结构或那些通过例如氨基酸中突变对催化活性，稳定性或热稳定性所进行的修饰的突变体。
Bst DNA聚合酶
Bca(exo-)DNA聚合酶
DNA聚合酶I克列诺(Klenow)片段
Vent DNA聚合酶
Vent(Exo-)DNA聚合酶(缺少核酸外切酶活性的Vent DNA聚合酶)
Deep Vent DNA聚合酶
Deep Vent(Exo-)DNA聚合酶(缺少核酸外切酶活性的Deep Vent DNA聚合酶)
Φ29phage DNA聚合酶
MS-2phage DNA聚合酶
OmniAmp DNA聚合酶
这些酶中，Bst DNA聚合酶、Bca(exo-)DNA聚合酶、OmniAmp DNA聚合酶是特别所需的酶，因为它们具有某种程度的热稳定性和高催化活性。在优选的实施方案中，本发明的反应可等温的实现，但由于解链温度的调节(Tm)等，不可能总是能利用所需温度条件来维持酶的稳定。因此，它是酶的热稳定所需的条件之一。尽管等温反应是可行的，热变性可提供核酸作为最初的模板，在这方面，热稳定酶的使用拓宽了试验方案的选择。
本发明合成或扩增核酸所必需的各种试剂可被预先包装，并以试剂盒的形式提供，具体地，本发明所提供的试剂盒包含作为合成互补链合成的引物和用于置换反应的外引物所必需的各种寡核苷酸，用于互补链合成的底物dNTP，用于实现链置换型互补链合成的DNA聚合酶，为酶反应提供合适条件的缓冲液，和用于检测合成反应产物所必需的介质。具体地，本发明优选的模式中，反应期间无需加入的试剂，并由此对于移入反应容器后一个反应所必需供给的试剂，其中仅通过加入样品就可启动该反应。通过利用可见光信号或荧光信号可在容器内检测反应产物的系统。反应后不必打开和关闭容器。这对于预防污染是非常有利的。
本发明合成具有竞争性茎环结构的核苷酸序列的单链核酸。该核酸具有例如下面的用途：第一特征是利用一分子中具有互补序列的特定结 构带来的优势，该特征可能利于检测，即有已知用于检测核酸的系统，其中其变化的信号取决于与互补核苷酸序列碱基配对。例如，通过结合使用双链特异性嵌入剂作为如上所述的检测试剂的方法，充分利用本发明合成产物特征的检测系统可得以实现。如果本发明合成反应的产物在所述检测系统发生一次热变性，并且返回到原始温度，分子内退火优先发生，并因此容许互补序列之间快速碱基配对。如果存在非特异性产物，分子中它们没有互补序列从而使通过热变性分离成2个或更多的分子后，它们不能立刻就返回到原始双链。通过在检测前提供的热变性步骤，伴随非特异反应的干扰得以降低。如果所用的DNA聚合酶不抗热，热变性步骤有反应终止的意思，并因此有利于控制反应温度。
第二特征是常常形成能碱基配对的竞争性形成发卡环(即茎环结构)。能碱基配对的竞争性发卡环的结构在图3中显示。如图3中看到的该环由核苷酸序列(从3’至5’端)F1c，N，R1，Nc构成，可进行分子内退火形成发卡环。
根据本发明优选的模式，在单链核酸中供给大量能碱基配对的环。这意味着大量的探针可与一分子核酸杂交以容许高灵敏度的检测。因此不仅可能实现改进灵敏度还可能实现基于特殊反应原理例如聚集作用来检测核酸的方法。例如，将固定在精细颗粒例如聚苯乙烯乳胶上的探针加入到本发明反应产物中，观察乳胶颗粒的聚集作用为产物与探针杂交。聚集作用的强度通过光学测定就可进行高灵敏度和定量观察。或者还可通过裸眼观察聚集作用，故还可建立不用光学的测定装置的反应系统。
此外，本发明反应产物允许一些可结合的标记，其中每核酸分子可进行层析检测。在免疫测定领域里，实际所应用是利用可见的检测标记使用层析介质的分析方法(免疫层析)。该方法基于分析物夹在固定于层析介质上的抗体和标记抗体间的原理，未反应的标记成分被洗脱。本发明的反应产物使该原理应用到核酸分析上。也就是，制备针对环部分的标记探针并固定在层析介质上为捕获准备捕获探针，以允许在层析介质里进行分析。序列与环部分互补的捕获探针得以利用，由于本发明的反 应产物具有大量的发卡环，产物与大量标记的探针结合以给出肉眼可识别信号。
本发明反应产物常常能供给碱基配对的环区，能拓宽其它各种检测系统。例如，利用表面胞质基因组使用固定探针检测该环部分的系统是可行的。此外，如果用双链特异嵌入物标记该环部分的探针，就可进行更灵敏的荧光分析。或积极利用本发明合成核酸的能力在3’-和5’-侧以形成能碱基配对的发卡。例如，设计一个环使其在正常型和不正常型间有共同的核苷酸序列，而设计其它环使其在其中产生差异。通过探针证实共同部分存在基因，而在其它区证实有不正常存在时，有可能组成特征分析系统。因为本发明合成核酸的反应也能等温的进行，值得一提的优点是，通过一般荧光光度计可进行实时(real-time)分析。直到此时，同一链中要退火的核酸的结构是已知的。然而，通过本发明获得的具有首尾退火成环的序列单链里的核酸是新的，它包含大量的能与其它碱基配对的环。
另一方面，通过本发明反应产物所给的大量的环自身能被用作探针，例如，在DNA芯片里，探针在有限的区域内高密度堆积，而该技术中可固定在某区域寡核苷酸数量有限，因此通过利用本发明产物大量能退火的探针可被高密度固定，即本发明的反应产物在DNA芯片上可用作固定的探针，扩增后反应产物可通过本领域已知的任何技术得以固定，或用固定的寡核苷酸作为本发明扩增反应的寡核苷酸，导致生成固定反应产物。因此通过使用固定的探针，大量样品DNA在有限的区域内得以杂交，结果预计可得到高信号值。
附图说明
图1是本发明核酸合成方法的步骤图解。
图2是本发明中第二核酸合成过程的步骤图解。
图3是本发明单链核酸所形成的竞争性发卡结构及后续扩增反应的示意图。
图4是本发明中核酸后续扩增反应的示意图。
图5是本发明合成方法形成的理想扩增产物的示意图。
图6是本发明实施例1中PH5SR靶核苷酸序列中对应的每个核苷酸序列区的位置关系。
图7是本发明实施例1在引物的作用下，PH5SR靶核苷酸序列DNA扩增过程中的实时荧光曲线图。
图8是本发明实施例2MERS-orf1a靶核苷酸序列中对应的每个核苷酸序列区的位置关系。
图9是本发明实施例2中以MERS-orf1a为模板以本发明单链核酸的合成方法获得的产物琼脂糖电泳结果的照片；其中，泳道1：碧云天O0107DNA Ladder；泳道2：1fmol MERS-orf1a dsDNA。
图10是本发明实施例3中限制酶消化产物的琼脂糖凝胶电泳结果的照片，其中所述产物是通过本发明核酸合成反应在实施例2中得到的。其中，
泳道1：纯化产物的XhoI消化
泳道2：纯化产物的HindIII消化
泳道3：纯化产物的XhoI与HindIII联用消化
泳道4：纯化产物
泳道5：分子量标记DNAladder。
图11是本发明实施例4中在引物的作用下，MERS-orf1a靶核苷酸序列DNA扩增过程中的实时荧光曲线图。
图12是本发明实施例5中在引物的作用下，MERS-orf1a靶核苷酸序列体外转录的RNA扩增过程中的实时荧光曲线图。
图13是本发明实施例6中在添加有加速探针作用下，对于靶核苷酸的扩增原理作用位点的示意图。
图14是本发明实施例6中在不同组合的加速探针引物组合的作用下，对于MERS-orf1a体系扩增的荧光强度随反应时间变化的曲线图。
图15是本发明实施例7中在设计的甲型流感病毒H1靶核苷酸引物的作用下，含甲型流感病毒H1基因靶核苷酸序列DNA扩增过程中的实时荧光曲线图。
图16是本发明实施例8中在鲤科疱疹病毒III型靶核苷酸引物的作用下，含鲤科疱疹病毒III型靶核苷酸序列DNA扩增过程中的实时荧光曲线图。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法，具体可参照《分子克隆实验指南》(第三版)J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行；下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1Pangasianodon hypophthalmus5S rRNA片段的扩增
本发明中所提出的恒温条件下合成核酸的方法，最小的目标片段可仅为60个碱基的核酸序列。在实施例中我们选用文献(Food Chem.2011；129:1860-4.)中的水晶巴丁鱼(Pangasianodon hypophthalmus)的5S rRNA基因(Genbank：HQ681111)作为目标基因，应用本发明方法实现了对该目标基因的恒温扩增。
本发明具有互补链以螺旋环的形式连接到单链里的核酸是利用人造的设计插入有酶切位点的Pangasianodon hypophthalmus5S rRNA(简称为PH5SR)(来自于GenBank:HQ681111)为模板尝试的。实验中用到两种引物即PH5SRNF(核苷酸序列如SEQ ID NO.l所示)和PH5SRNR(核苷酸序列如SEQ ID NO.2所示)。通过利用临近堆积现象将这些设计成退火成环的区。此外，将这些引物设置为高浓度使PH5SRNF(或PH5SRNR)的退火优先发生。
靶核苷酸序列每个区的位置关系在图2中显示。通过所述引物PH5SRNF和PH5SRNR，在靶核苷酸PH5SR两端合成两个Nc区段与靶核苷酸序列上的N区段竞争形成发卡环，合成过程参见图2所示。通过这些引物合成本发明核酸的方法的反应溶液的组合如下面所示。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
引物：
1600nM PH5SRNF
1600nM PH5SRNR
靶核酸:PH5SR dsDNA(核苷酸序列如SEQ ID NO.3所示)。参见图6，为PH5SR靶核苷酸序列中对应的每个核苷酸序列区的位置关系。
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。荧光强度随反应时间变化的曲线如图7所示。将荧光检测应用于其中可实现实时监测的目的，通过实时扩增曲线可提前判断结果。
实施例2对于MERS-orf1a中片段的扩增
本发明具有互补链以螺旋环的形式连接到单链里的核酸是利用人造的设计插入有酶切位点的MERS-orf1a(来自于GenBank:KX108946.1)为模板尝试的。实验中用到两种引物为Mo1aNF(核苷酸序列如SEQ ID NO.4所示)和Mo1aNR(核苷酸序列如SEQ ID NO.5所示)。通过利用临近堆积现象将这些设计成退火成环的区。此外，将这些引物设置为高浓度使Mo1aNF(或Mo1aNR)的退火优先发生。
通过所述引物Mo1aNF和Mo1aNR，在靶核苷酸MERS-orf1a两端合成两个Nc区段与靶核苷酸上的N区段竞争形成发卡环。通过这些引物合成本发明核酸的方法的反应溶液的组合在下面所示。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
引物：
1600nM Mo1aNF
1600nM Mo1aNR
靶核酸:MERS-orf1a dsDNA(核苷酸序列如SEQ ID NO.6所示)。参见图8，为MERS-orf1a靶核苷酸序列中对应的每个核苷酸序列区的位置关系。
混合物于63℃反应1小时，反应后，于80℃10分钟终止该反应，然后重新转到用冰预冷的水中。
反应的证实：将1μL常规核酸电泳上样缓冲液(碧云天DNA ladder附赠)加到5μL上面的反应溶液中，样品于90mV在GelRed预染的(Biotum)1％琼脂糖凝胶(TAE溶解)电泳1小时。用碧云天O0107DNA Ladder作为分子量标记。电泳后的凝胶以验证核酸。结果在图9中显示，获得了宽分子量分布的核酸产物，即验证了发明方法所得到的核酸可无限自组装退火延伸得到超大核酸分子。
实施例3通过限制酶的消化证实反应产物
为了阐清具有互补核苷酸序列以环状结构连接在单链内的本发明实施例2获得的核酸结构，用限制酶消化产物。如果通过消化能生成理论上的片段，同时不存在(disappear)如实施例2中观察到的高分子量处产生不清晰成片条带模式和未被电泳的带，就可预计任何这些产物为本发明具有互补序列交替地连接在单链内的核酸。
实施例2中反应溶液通过用酚处理及乙醇的沉淀作用得以沉积和纯化，回收产生的沉淀并重新溶于超纯水中，用限制酶HindIII、XhoI及此二酶联用于37℃消化2小时，样品于90mV在GelRed预染的(Biotum) 1％琼脂糖凝胶(TAE溶解)电泳1小时。用碧云天O0107DNA Ladder作为分子量标记。电泳后的凝胶以验证核酸。结果在图10中显示，所获得的核酸产物由大片段可酶切为小片段，证明产物为针对目标核酸扩增所得，未出现非特异性扩增，证实了本发明方法的特异性。
实施例4应用EvaGreen验证反应产物
EvaGreen同SYBR Green I类似，是一种结合于所有dsDNA双螺旋小沟区域的具有绿色激发波长的染料，其对PCR等核酸扩增反应的抑制远小于后者。在游离状态下，EvaGreen发出微弱的荧光，但一旦与双链DNA结合后，荧光大大增强。因此，EvaGreen的荧光信号强度与双链DNA的数量相关，可以根据荧光信号检测出核酸扩增体系存在的双链DNA数量。
通过引物Mo1aNF(核苷酸序列如SEQ ID NO.4所示)和Mo1aNR(核苷酸序列如SEQ ID NO.5所示)，合成本发明核酸的方法的反应溶液的组合如下面所示。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
引物：
1600nM Mo1aNF1600nM Mo1aNR
靶核酸:MERS-orf1a dsDNA(核苷酸序列如SEQ ID NO.6所示)。设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。 荧光强度随反应时间变化的曲线如图11所示。将荧光检测应用于其中可实现实时监测的目的，通过实时扩增曲线可提前判断结果。
实施例5应用基于EvaGreen的实时荧光实现RNA靶基因扩增
AMV反转录酶可以RNA为模板合成cDNA，配合Bst DNA聚合酶可以实现RNA的检测。
通过引物Mo1aNF(核苷酸序列如SEQ ID NO.4所示)和Mo1aNR(核苷酸序列如SEQ ID NO.5所示)以RNA为模板合成cDNA，反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
5U AMV反转录酶
1X EvaGreen(Biotum)
引物：
1600nM Mo1aNF
1600nM Mo1aNR
靶核酸:MERS-orf1a RNA(RNA核酸序列如SEQ ID NO.7所示)。该MERS-orf1a RNA是由MERS-orf1a(序列如SEQ ID NO.6所示)体外转录而得。
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。荧光强度随反应时间变化的曲线如图12所示。此结果说明本方法应用于RNA检测同样可行。
实施例6应用加速探针对于MERS-orf1a dsDNA靶基因扩增
通过这些引物合成本发明核酸的方法的反应溶液的组合在下面所示。
将加速探针组合分为四组，仅引物组合不同(其中加速探针1指包含引物F2和R2，加速探针2指包含引物Fin和Rin)：
a，无加速探针1、无加速探针2
b，有加速探针1、无加速探针2
c，无加速探针1、有加速探针2
d，有加速探针1、有加速探针2
参见图13，该图为在添加有加速探针作用下，对于靶核苷酸的扩增原理作用位点的示意图。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
a引物：
1600nM Mo1aNF(序列如SEQ ID NO.4所示)
1600nM Mo1aNR(序列如SEQ ID NO.5所示)
b引物：
1600nM Mo1aNF(序列如SEQ ID NO.4所示)
1600nM Mo1aNR(序列如SEQ ID NO.5所示)
200nM Mo1aF2(序列如SEQ ID NO.8所示)
200nM Mo1aR2(序列如SEQ ID NO.9所示)
c引物：
1600nM Mo1aNF(序列如SEQ ID NO.4所示)
1600nM Mo1aNR(序列如SEQ ID NO.5所示)
800nM Mo1aFin(序列如SEQ ID NO.10所示)
800nM Mo1aRin(序列如SEQ ID NO.11所示)
d引物：
1600nM Mo1aNF(序列如SEQ ID NO.4所示)
1600nM Mo1aNR(序列如SEQ ID NO.5所示)
200nM Mo1aF2(序列如SEQ ID NO.8所示)
200nM Mo1aR2(序列如SEQ ID NO.9所示)
800nM Mo1aFin(序列如SEQ ID NO.10所示)
800nM Mo1aRin(序列如SEQ ID NO.11所示)
a，b，c，d各组引物对应的靶核酸均为:MERS-orf1a dsDNA(序列如SEQ ID NO.6所示)。设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。荧光强度随反应时间变化的曲线如图14所示。比较a组与b组的Ct值，说明加速探针1起到加速效果；比较a组与c组的Ct值，说明加速探针2起到加速效果；同时比较a组与b组、c组的Ct值，说明加速探针2起到加速效果好于加速探针1；同时比较a组、b组、c组c组与的Ct值，说明加速探针1与加速探针2相互配合起到协同作用。
实施例7对于甲型流感病毒H1dsDNA靶基因扩增
甲型H1N1病毒属于正粘病毒科(0rthomyxoviridae)，甲型流感病毒属(Influenza virus A)，甲型H1N1流感症状与感冒类似，患者会出现发烧、咳嗽、疲劳、食欲不振等。2009年H1N1曾大面积流行，引起了一定程度的恐慌。因H1N1的核酸检测一般采用逆转录后应用PCR技术检测cDNA。故应用本发明方法设计出新的引物亦可应用于H1N1病毒的检测。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
引物：
1600nM H1-NF(序列如SEQ ID NO.12所示)
1600nM H1-NR(序列如SEQ ID NO.13所示)
靶核酸为:H1dsDNA(序列如SEQ ID NO.14所示)
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。荧光强度随反应时间变化的曲线如图15所示。扩增曲线说明本方法可适用于甲型流感病毒的检测应用领域。
实施例8对于鲤科疱疹病毒III靶基因扩增
鲤科鱼类疱疹病毒感染所导致的锦鲤疱疹病毒病、疱疹病毒性造血器官坏死病、鲤痘疮病严重威胁鲤科鱼类养殖。该病毒所具有极高致病性、极强传染性的特点，造成该病在世界范围流行，感染鱼类的死亡率可达80％～100％。该病已引起国际动物卫生组织(OIE)的高度关注，将其列为重点疫病目录，同时我国也将该病列为二类动物疫病，并已开展日常的监测工作。开发相应的检测技术实现相关疫病的快速检测以及时应对疫情则显得十分重要。故挑选鲤科疱疹病毒III应用本发明方法作为潜在应用对象。
反应溶液组合如下，其余用ddH2O至25μL
20mM Tris-HCl pH8.8
10mM KCl
10mM(NH4)2SO4
14mM MgSO4
0.1％Triton X-100
1M甜菜碱
1.25mM dNTP
8U Bst DNA聚合酶(NEW ENGLAND Biolabs)
1X EvaGreen(Biotum)
引物：
1600nM CyHVIII-NF(序列如SEQ ID NO.15所示)
1600nM CyHVIII-NR(序列如SEQ ID NO.16所示)
靶核酸为:CyHVIII dsDNA(序列如SEQ ID NO.17所示)
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为60min。荧光强度随反应时间变化的曲线如图16所示。扩增曲线说明本方法可适用鲤科疱疹病毒等水产防控的检测应用领域。
上述仅为本发明的部分优选实施例，本发明并不仅限于实施例的内容。对于本领域中的技术人员来说，在本发明技术方案的构思范围内可以有各种变化和更改，所作的任何变化和更改，均在本发明保护范围之内。
Figure PCTCN2017101789-appb-000001
Figure PCTCN2017101789-appb-000002
Figure PCTCN2017101789-appb-000003
Figure PCTCN2017101789-appb-000004
Figure PCTCN2017101789-appb-000005
Figure PCTCN2017101789-appb-000006
Figure PCTCN2017101789-appb-000007
Figure PCTCN2017101789-appb-000008

Claims (10)

  1. 一种恒温条件下合成核酸的方法，包括以下步骤：
    1)提供一种核酸，所述核酸的5’末端具有可与同一条链上的N区退火的Nc区，同时该核酸的3’末端具有可与同一条链上的N区退火的Nc区，此核酸的5’末端和3’末端所具有的Nc区与同一链上的N区退火存在竞争关系；
    2)以步骤1)所述核酸为模板，将已与N区退火的Nc区的3’端为合成起点，合成其自身的互补核酸链；
    3)通过聚合酶催化链置换型互补链合成反应进行互补链合成，以置换步骤2)中所合成的互补核酸链。
  2. 根据权利要求1所述的一种恒温条件下合成核酸的方法，其特征在于，具体包括以下步骤：
    1)提供一种核酸的步骤，该核酸的5’末端和3’末端均具有能够与同一条链上的N区退火的Nc区；所述3’末端的Nc区与N区退火时能形成环，所述环包含能进行碱基配对的F1c区；所述5’末端的Nc区与N区退火时能形成环，所述环包含能进行碱基配对的R1区；所述核酸的5’末端和3’末端所具有的Nc区与同一链上的N区的退火存在竞争关系；
    2)使第一寡核苷酸I与步骤1)提供的所述核酸的F1c区退火，然后以所述第一寡核苷酸I的F1区作为合成起点，进行合成步骤；其中所述第一寡核苷酸I包括N区与Fl区；
    3)以步骤1)提供的所述核酸为模板合成其自身的互补链，将已与N区退火的Nc区的3’端为合成起点；将合成完后的核酸序列称为核酸A；
    4)使第二寡核苷酸II与步骤3)提供的所述核酸A的R1c区退火，然后以所述第二寡核苷酸II的R1区作为合成起点，进行合成步骤；其中所述第二寡核苷酸II包括R1区和Nc区；
    5)以步骤3)提供的所述核酸A为模板合成其自身的互补链，将3’端已与Nc区退火的N区为合成起点，获得在其一条链上具有首尾互补核苷酸序列的核酸链，并且该核酸链上具有交替连接的互补核苷酸序列区。
  3. 根据权利要求1所述的恒温条件下合成核酸的方法，其特征在于，步骤1)所述核酸的制备方法，包括以下步骤：
    1-1)退火步骤，使第一寡核苷酸I与模板的F1c区退火，其中所述模板的3’末端包括F1c区和位于F1c区5’侧的N区，该模板的5’末端包括R1区， 其中所述第一寡核苷酸I包括N区与Fl区，所述N区与F1区的5’侧相连，其中，
    F1区：具有与F1c区互补的核苷酸序列的区，
    N区：与Nc区互补的核苷酸序列的区；
    1-2)以所述第一寡核苷酸I的F1区作为合成起点，合成第一核酸；所述第一核酸具有与所述模板互补的核苷酸序列，所述第一核酸的5’末端具有可与同一条链上的Nc区退火的N区，并且通过所述Nc区与N区的退火可形成茎环；
    1-3)利用聚合酶催化链置换反应置换获得步骤1-2)所合成的第一核酸；
    1-4)退火步骤，使第二寡核苷酸II与步骤1-3)所得第一核酸的R1c区退火，其中所述第二寡核苷酸II包括R1区和Nc区，并且Nc区与R1区的5’侧相连；其中，
    R1区：具有与R1c区互补的核苷酸序列的区，
    Nc区：与N区互补的核苷酸序列的区；
    1-5)以所述第二寡核苷酸II的R1区作为合成的起点，合成第二核酸；
    1-6)利用聚合酶催化链置换反应置换所述第二核酸获得步骤1)所述的核酸。
  4. 根据权利要求3所述的恒温条件下合成核酸的方法，其特征在于：步骤1-1)所述模板为RNA，步骤1-2)中的第一核酸通过具有反转录酶活性的酶来合成。
  5. 根据权利要求2-4任一项所述的恒温条件下合成核酸的方法，其特征在于：所述F1c区、N区和R1区的核酸片段均为15-60bp。
  6. 根据权利要求1或2所述的恒温条件下合成核酸的方法，其特征在于：所述获得的核酸链能够自主配对无限延伸，该核酸链上3’端的Nc区会与该链上的互补区段N区配对作为合成起点以自身为模板使所述核酸链不断延伸。
  7. 根据权利要求2所述的恒温条件下合成核酸的方法，其特征在于：所述合成核酸的方法中通过引入加速引物X2和/或Xin的方法使得核酸扩增加速进行；其中X2是位于原始核酸互补链的F1区及R1区的5’侧的区段，Xin是位于F1c区到N区及Nc区到R1c的中间区段。
  8. 一种合成核酸的试剂盒，所述核酸在其一条链上具有首尾互补核苷酸序列，并且该核酸链上具有交替连接的互补核苷酸序列；所述试剂盒包括以 下组分：
    第一寡核苷酸I，其包括F1区和N区，所述N区与F1区的5’侧相连，其中，
    F1区：具有与F1c区互补的核苷酸序列的区，和
    N区：与Nc区互补的核苷酸序列的区；
    第二寡核苷酸II，其包括R1区和Nc区，所述Nc区与R1区的5’侧相连，其中，
    R1区：具有与R1c区互补的核苷酸序列的区，和
    Nc区：与N区互补的核苷酸序列的区；
    催化链置换型互补链合成反应的DNA聚合酶；
    核苷酸，其作为所述DNA聚合酶的底物。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述试剂盒还包括加速引物X2和/或Xin，其中X2是位于原始核酸互补链的F1区及R1区的5’侧的区段，Xin是位于F1c区到N区及Nc区到R1c的中间区段。
  10. 权利要求8或9所述试剂盒在合成核酸或检测样品中靶核苷酸序列中的应用。
PCT/CN2017/101789 2017-09-14 2017-09-14 一种恒温条件下合成核酸的方法及试剂盒 WO2019051732A1 (zh)

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