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WO2018132939A1 - 一种恒温条件下合成核酸的方法 - Google Patents

一种恒温条件下合成核酸的方法 Download PDF

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WO2018132939A1
WO2018132939A1 PCT/CN2017/071394 CN2017071394W WO2018132939A1 WO 2018132939 A1 WO2018132939 A1 WO 2018132939A1 CN 2017071394 W CN2017071394 W CN 2017071394W WO 2018132939 A1 WO2018132939 A1 WO 2018132939A1
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nucleic acid
template
complementary
synthesizing
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French (fr)
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杜昱光
毛瑞
刘洪涛
王倬
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Institute of Process Engineering of CAS
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Institute of Process Engineering of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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

Definitions

  • 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.
  • SNPs single nucleotide polymorphisms
  • 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.
  • HSD 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.
  • 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 10 9 -10 10 times amplification in 15-60 minutes, and the reaction can produce a large amount of amplification product, namely magnesium pyrophosphate white precipitate. The presence or absence of white precipitate can be visually observed to determine whether the target gene exists.
  • Japan Rongyan Company has also developed a turbidity meter to achieve real-time monitoring of amplification reactions. In addition to high specificity and high sensitivity, the LAMP method is very simple to operate.
  • RPA Recombinase Polymerase Amplification
  • PSR polymerase helix reaction
  • the introduction of foreign genes into the primer sequences of this method will greatly increase the likelihood of mismatches leading to erroneous results.
  • the closed loop structure in the closed structure in the initial structure is only about 20 base pairs, which makes the intermolecular hydrogen bond weak and makes the spiral loop unstable, and if the length of the foreign gene is too long, Affect the spiral ring formation process.
  • 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 and the LAMP and PSR methods.
  • the primer working mode is redesigned and planned based on PCR primers, and the amplification of the helical loop is initiated. Structure, designing the formation process of the structural loop, and having a new feature of overlapping bases with more base pairs, no introduction of foreign gene fragments, and amplification products can be reused. 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 .
  • 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, LAMP, and PSR.
  • 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. . That is, 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 to which the method is applied.
  • the method for synthesizing nucleic acid under the constant temperature condition of the invention comprises the following steps:
  • step 2) synthesizing its own complementary strand with the nucleic acid of step 1) as a template, the R1rc region of the complementary strand is annealed to the R1 region, and the 3' end of the F1r region annealed in the Flc region constitutes a helical loop as a starting point for synthesis;
  • step 3 3) performing complementary strand synthesis by polymerase catalytic strand displacement-type complementary strand synthesis reaction to replace the complementary strand synthesized in step 2), wherein the polynucleotide comprises one at its 3' end and in step 2) A sequence complementary to any region of the complementary complementary strand.
  • the method for synthesizing a nucleic acid according to the present invention comprises the steps of:
  • an annealing step of annealing the first oligonucleotide I to the F1c region of the template wherein the 3' end of the template comprises an F1c region and an F2c region located on the 3' side of the F1c region, the 5' end of the template comprising the R1 region And an R2 region located on the 5' side of the R1 region, wherein the first oligonucleotide I includes an R1r region and an F1 region, and the Rlr region is connected to the 5' side of the F1 region, wherein
  • F1 region a region having a nucleotide sequence complementary to the template F1c region
  • R1r area an area opposite to the template R1 area
  • a step of synthesizing a first nucleic acid having a nucleotide sequence complementary to the template the step of synthesizing a first nucleic acid using the F2 region of the first oligonucleotide I as a starting point for synthesis,
  • the 3' end of the first nucleic acid has an F1 region which can anneal to a portion of the F1c region on the same chain, and a helical ring which can be formed by simultaneous annealing of the F1 region and the Flc region;
  • step ii) replacing the first nucleic acid synthesized in step ii) with a polymerase catalytic strand displacement reaction, wherein the first outer primer I annealed to the F2c region on the 3' side of F1c in the template is used as a starting point for synthesis, and
  • step iii) an annealing step of annealing the second oligonucleotide II to the R1c region of the first nucleic acid obtained in step iii), wherein the second oligonucleotide II comprises the R1 region and the F1r region, and the Flr region and the R1 region The 5' side is ligated, and the second oligonucleotide II is the reverse sequence of the first oligonucleotide I;
  • R1 region a region having a nucleotide sequence complementary to the R1c region of the first nucleic acid
  • Flr region a region opposite to the F1 region of the first nucleic acid
  • a second external primer II which anneals to the R2c region on the 3' side of R1c in the first nucleic acid serves as a starting point for the synthesis.
  • the template of step i) is RNA
  • 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 RNA of various viruses and the like.
  • RNA detection for the MERS-CoV virus such as: orf1a, orf1b segment of the RNA.
  • the sequence of the oligonucleotide used in the F1 region and the R1r region, or the sequence connecting the R1 region and the F1r region may be none, but if a linker (L) is introduced,
  • the sequence must be a reverse sequence, ie, the two oligonucleotides are 5'-R1r-L-F1-3', and 5'-F1r-Lr-R1-3' must be a reverse complement sequence (L and Lr)
  • R1r is the reverse sequence of R1
  • F1r is the reverse sequence of F1
  • linker is generally a restriction endonuclease sequence.
  • a method of synthesizing a nucleic acid according to the present invention, wherein the synthetic nucleic acid is in one of its chains A nucleic acid having a first-tail complementary nucleotide sequence.
  • 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 constant temperature of 60 to 65 °C.
  • a method of synthesizing a 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: (external primer or template 3' side Region) ⁇ (F2c or F2, and, R2c or R2) ⁇ (F1c or F1, and, Rlc or Rl).
  • the obtained second nucleic acid can be accelerated by introducing a method of accelerating the probe Xin, wherein Xin is an intermediate portion located in the F1 region to the R1 region.
  • the present invention can repeat steps 1) to 3) to synthesize long-chain nucleic acids using the complementary strand after the replacement of step 3).
  • 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 allowed to be 0.2 to 3.0M.
  • kit for synthesizing nucleic acid under constant temperature conditions of the invention characterized in that the kit comprises:
  • An oligonucleotide I comprising an F1 region and an R1r region, wherein the R1r region is linked to the 5' side of the F1 region, wherein
  • F1 region a region having a nucleotide sequence complementary to the F1c region of the template
  • R1r zone a zone opposite to the R1 zone of the template
  • An oligonucleotide II comprising an R1 region and an F1r region, wherein the F1r 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 of the template
  • F1r the area opposite to the F1 area of the template
  • a first primer I having a nucleotide sequence complementary to the F2c region on the 3' side of the F1c region of the nucleic acid as a template
  • a second primer II having a nucleotide sequence complementary to the R2c region on the 3' side of the R1c region of the nucleic acid as a template
  • Nucleotide which serves as a substrate for the DNA polymerase.
  • kit according to the present invention, wherein the kit further comprises a detection reagent for detecting a nucleic acid synthesis reaction product.
  • kit further comprises an acceleration probe Xin, wherein Xin is a nucleotide segment located in the middle segment of the F1 region to the R1 region of the template, specifically, the Fin and Rin regions located between F1 and R1.
  • Xin is a nucleotide segment located in the middle segment of the F1 region to the R1 region of the template, specifically, the Fin and Rin regions located between F1 and R1.
  • 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 helical loop is added to the above amplification product, and hybridization between the two is 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 having a complementary nucleotide sequence of the first and the tail is the object of the present invention, and the nucleic acid refers to a nucleic acid having a target nucleic acid sequence which is ligated side by side in a single strand with mutually complementary nucleotide sequences.
  • a nucleotide sequence for helixing between complementary strands should be included in the present invention. This sequence is referred to as a helical loop sequence in the present invention.
  • the nucleic acids synthesized by the present invention consist essentially of mutually complementary strands joined by a helical loop sequence.
  • a strand that cannot be separated into two or more molecules when paired bases are separated is referred to as a single strand, whether or not partially involved in base pairing.
  • 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 complementary nucleotide sequence end-to-end linked 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 an annealing product thereof in a curved portion
  • a loop that does not involve base pairing is constructed.
  • 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 be used as a template. To synthesize the starting point of the complementary strand.
  • 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. Therefore, even if the nucleic acid strands constituting the base pairing are single-stranded, 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 having the head-to-tail complementary loop-forming sequence synthesized by the present invention may not have the same structure as the naturally occurring nucleic acid, and it is generally known that when a nucleic acid is synthesized by a nucleic acid polymerase, a nucleotide derivative is used as a nucleotide derivative.
  • a substrate can synthesize a nucleic acid derivative.
  • 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 product nucleic acid may be a fluorescent derivative.
  • the product may be DNA or RNA.
  • the resulting product is determined by a combination of a primer structure for realizing the polymerization of the nucleic acid, a type of the polymerization substrate, and a reagent for the polymerization.
  • the synthesis of a nucleic acid having the above structure is carried out by using a DNA polymerase having a strand displacement activity and the F1r region having a portion of the Flc region on the same strand at the 3'-end to form a synthetic complementary strand.
  • a DNA polymerase having a strand displacement activity and the F1r region having a portion of the Flc region on the same strand at the 3'-end to form a synthetic complementary strand.
  • a hairpin loop is formed, the hairpin loop sequence itself is used as a template, and a spiral loop is formed, and the loop loop sequence itself is used as a template, and in the present invention, the head and tail portions of the loop are provided.
  • a region capable of base pairing and having new features for utilizing this region in the synthesis of complementary strands By using this region as a starting point for synthesis, the complementary strand previously synthesized with the helical loop sequence itself as a template was replaced.
  • nucleic acid is used in the present invention, and the nucleic acid of the present invention generally includes both DNA and RNA. However, nucleic acids or modified nucleotides derived 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 nucleic acid range of the present invention. Typically, 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. For example, 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 F1r region is provided at the 3'-end, and can be annealed with a portion of Flc on the same chain, and the F1r region is annealed with Flc on the same chain to form an R1 region including base pairing.
  • the loop can be obtained in a variety of ways.
  • 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.
  • the complementary sequence is a sequence that can be annealed under stringent conditions, providing a 3'-end as a 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 phosphorus
  • the acid diester bond is a linkage. 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. The separated fraction can be detected by nucleic acid specific indicators or by hybridization with a labeled probe.
  • 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 composed only by adding a reverse transcriptase, that is, using RNA as a template, annealing of F1 and F1c in the template by reverse transcriptase is possible to synthesize a complementary strand and from annealing to F2c.
  • Primer F2 synthesizes the complementary strand for the synthetic starting point and simultaneously replaces the previously synthesized complementary strand, and the outer primer F2 is located on the 3'-side of F1c.
  • 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 first single-stranded nucleic acid from the RNA is passed through the same enzyme. The synthesis, and then the DNA-templated reaction 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, and an appropriate amount of salt, KCl, NaCl, (NH4) 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 white. Protein 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 or Bca (exo-) DNA polymerase are particularly desirable enzymes because they have some 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 in which the head-to-tail sequence can be annealed to a loop.
  • the nucleic acid has, for example, the following utility:
  • the first feature is the advantage of utilizing a specific structure having a complementary sequence in one molecule, which may facilitate detection, ie, a system known to detect nucleic acids, wherein the signal of the change depends on Base pairing with a complementary nucleotide sequence.
  • 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 the often closed loop that forms a base-paired end-to-end linkage.
  • the structure of the base-pairable loop is shown in Figure 3.
  • the loop consists of nucleotide sequences F1, R1, F1c, R1c which can be subjected to intramolecular annealing to form a closed loop.
  • a large number of base-pairable loops are supplied in a single-stranded nucleic acid.
  • 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 whose sequence is complementary to a loop moiety is utilized. Since the reaction product of the present invention has a large number of loops, the product combines 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 is on the 3'- and 5'- sides to form base-pairing helical loops. 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 graphical representation of the steps of the synthesis of a first nucleic acid of the invention.
  • Figure 2 is a graphical representation of the steps of the synthesis of a second nucleic acid of the invention.
  • Figure 3 is a diagram showing the structure of a ring formed by the single-stranded nucleic acid of the present invention.
  • Figure 4 is a diagram showing the acceleration of the loop structure formed by the single-stranded nucleic acid of the present invention by the addition of additional primers.
  • Figure 5 is a graph showing the positional relationship of each nucleotide sequence constituting an oligonucleotide in the target nucleotide sequence of MERS-orf1b.
  • Fig. 6 is a photograph showing the results of agarose electrophoresis of a product obtained by a method of synthesizing a single-stranded nucleic acid of the present invention using MERS-orf1b as a template.
  • Figure 7 is a graph showing the positional relationship of each nucleotide sequence constituting an oligonucleotide in the target nucleotide sequence of MERS-orf1b.
  • Fig. 8 is a photograph showing the results of agarose gel electrophoresis of a restriction enzyme digestion product obtained in Example 1 by the nucleic acid synthesis reaction of the present invention. among them,
  • Lane 1 Biyuntian O0107DNA Ladder
  • Lane 2 1fmol MERS-orf1b dsDNA
  • Figure 9 is a graph showing the real-time fluorescence of an increase in DNA containing the MERS-orf1b target nucleotide sequence under the action of a primer.
  • Figure 10 is a graph showing the real-time fluorescence curve of the increase in DNA containing the MERS-orf1b target nucleotide sequence under the action of the primer.
  • Figure 11 is a graph showing the fluorescence intensity of the amplification system of different DNA target concentrations as a function of reaction time under the action of primers by the addition of accelerated primers.
  • Figure 12 is a schematic flow diagram of a method of synthesizing a nucleic acid of the present invention.
  • Figure 13 is a schematic representation of the helical structure of the nucleic acid synthesized in the present invention.
  • 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 MERS-orf1b (from GenBank: NM_001012270.1) as a template.
  • MERS-orf1b from GenBank: NM_001012270.1
  • Mo1bHF, Mo1bHR, Mo1bF2 and Mo1bR2 were used in the experiment.
  • Mo1bF2 and Mo1bR2 are external primers that replace the first nucleic acid obtained by using Mo1bHF and Mo1bHR as synthesis starting points, respectively. Because the external primers after synthesis by Mo1bHF (or Mo1bHR) are primers for the starting point of complementary strand synthesis. 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 Mo1bHF (or Mo1bHR).
  • nucleotide sequences constituting each primer are shown in the sequence listing, and the structural features of the primers are summarized below. Furthermore, the positional relationship for each region of the target nucleotide sequence is shown in FIG.
  • nucleic acid in which R1 and R1rc, F1c and F1r are complementary to a helical loop is synthesized.
  • the combination of the reaction solutions of the method of synthesizing the nucleic acid of the present invention by these primers is shown below.
  • SEQ ID NO. 1 GTACGAAGGGCATTACGCTCTCGTGTTATTTCCAGG;
  • SEQ ID NO. 2 GGACCTTTATTGTGCTCTCGCATTACGGGAAGCATG;
  • SEQ ID NO. 3 TACCCGCAAATGTCCCATA;
  • SEQ ID NO. 4 TGTAGAGGCACATTGGTG;
  • 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.
  • Example 2 confirmed the reaction product by restriction enzyme digestion
  • Example 1 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 a theoretical fragment is produced by digestion, and at the same time, there is no such phenomenon that the high molecular weight observed in Example 1 produces an unclear strip pattern and a band that is not electrophoresed, and 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 1 The reaction solution in Example 1 was deposited and purified by treatment with phenol and ethanol, and the resulting precipitate was recovered and redissolved in ultrapure water, digested with restriction enzyme HindIII at 37 ° C for 2 hours, and the sample was pretreated at 90 mV in GelRed.
  • the Biyuntian O0107 DNA Ladder was used as the molecular weight marker.
  • the gel after electrophoresis is used to verify the nucleic acid. The results are shown in Figure 7, with each lane being relative to the sample below.
  • 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.
  • AMV reverse transcriptase can synthesize cDNA using RNA as a template, and Bst DNA polymerase can detect RNA.
  • SEQ ID NO. 6 ACAGTTCCTGGATATCCTAAGCT;
  • SEQ ID NO. 7 ACAGCCTCTTCACGAGTAATG.

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Abstract

提供了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3'末端具有与同一条链上的F1c区退火的F1r区，并且通过所述F1r区与Flc区的同时退火可形成螺旋环；2）以步骤1）的核酸为模板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而进行互补链合成，以置换步骤2）中所合成的互补链。其中，所述寡核苷酸在引物的5'-侧所提供的核苷酸序列，基本上与以该引物为合成起点而合成的区域相同。

Description

一种恒温条件下合成核酸的方法 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。
当前人类基因组计划已经完成，单核苷酸多态性(SNPs)显示出数量多、分布广泛的特点，故其分析逐步受到重视。通过设计引物使其核苷酸序列包含SNPs，依靠PCR扩增来检测SNPs是可行的，即通过是否存在与引物互补的核苷酸序列可通过是否存在反应产物的决定得出推断。然而，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，HAD)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。
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，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。
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。
解放军军事医学科学院的刘威等(Liu，Dong et al.2015)提出了一种称为聚合酶螺旋反应(PSR)的新型核酸等温扩增技术，该反应使用与LAMP反应一样的酶，通过在设计的PCR引物的5’端引入特定序列进行扩增形成5’和3’端可以自组装形成闭合螺旋状的结构以提供3’-OH作为合成的起点，并以之为扩增起始结构，而后大量合成相同序列的DNA单链，最终形成长链的螺旋结构产物。但此方法的引物序列中引入了外源基因，这将极大增加错配的可能性而导致错误的结果。此外，起始结构中的闭合环状结构中重叠成环部分仅为20碱基对左右，此将分子间氢键较弱进而使得螺旋环不稳定，同时若外源基因的长度过长则将影响螺旋环形成过程。
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常见双螺旋结构及LAMP和PSR方法的启发的新原理，以PCR引物为基础重新设计并规划了引物工作模式，螺旋环扩增起始结构，设计该结构环的形成过程，并且具有重叠成环部分碱基对较多、不引入外源基因片段、扩增产物可重新利用的新特点。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和PSR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的试剂盒。
本发明的具有技术方案如下：
本发明的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3’末端具有可与同一条链上的F1c区退火 的F1r区，并且通过所述F1r区与Flc区的同时退火可形成螺旋环；
2)以步骤1)所述核酸为模板合成其自身的互补链，所述互补链的R1rc区与R1区退火，而Flc区退火的F1r区的3'末端组成螺旋环，作为合成起点；
3)通过聚合酶催化链置换型互补链合成反应而进行互补链合成，以置换步骤2)中所合成的互补链，其中所述多核苷酸在其3’末端包含一种与步骤2)中合成的互补链的任意区域互补的序列。
根据本发明所述的合成核酸的方法，其中，步骤1)所述核酸的制备方法，包括以下步骤：
i)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3’侧的F2c区，该模板的5’末端包括R1区和位于R1区5’侧的R2区，其中所述第一寡核苷酸I，包括R1r区与Fl区，所述Rlr区与F1区的5’侧相连，其中，
F1区：具有与模板F1c区互补的核苷酸序列的区，
R1r区：与模板R1区反向的区；
ii)合成第一核酸的步骤，所述第一核酸具有与所述模板互补的核苷酸序列，此步骤以第一寡核苷酸I的F2区作为合成的起点合成第一核酸，所述第一核酸的3'末端具有可与同一条链上的部分F1c区退火的F1区，并且通过所述F1区与Flc区的同时退火可形成的螺旋环；
iii)利用聚合酶催化链置换反应置换步骤ii)中合成的第一核酸，其中与模板中的F1c的3’侧的F2c区退火的第一外引物I用作合成的起点，和
iv)退火步骤，使第二寡核苷酸II与步骤iii)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F1r区，并且Flr区与R1区的5’侧相连，第二寡核苷酸II为第一寡核苷酸I的反向序列；其中，
R1区：具有与第一核酸的R1c区互补的核苷酸序列的区，
Flr区：与第一核酸的F1区反向的区；
v)以所述第二寡核苷酸II作为合成的起点，合成第二核酸，并利用聚合酶催化链置换反应置换该第二核酸获得步骤1)所述的核酸；其中，置换第二核酸时，与第一核酸中的R1c的3’侧的R2c区退火的第二外引物II用作合成的起点。
进一步地，步骤i)所述模板为RNA，步骤ii)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RNA，例如各种病毒的RNA等等。比如，用于MERS-CoV病毒的RNA检测，如：该RNA的orf1a，orf1b区段等。
本发明在形成起始螺旋结构的过程中，所使用的寡核苷酸中连接F1区与R1r区，或者连接R1区与F1r区的序列可以为无，但如若引入连接序列(linker，L)，该序列必须为反向序列，即两寡核苷酸为5’-R1r-L-F1-3’、5’-F1r-Lr-R1-3’中L和Lr须为反向互补序列(R1r为R1的反向序列，F1r为F1的反向序列)，linker一般为限制性内切酶位点序列。
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℃的恒定温度下进行合成。
根据本发明所述的合成核酸的方法，其中，每种寡核苷酸与其在模板中的互补区之间的解链温度在相同严谨条件下存在以下的关系：(外引物或模板3'侧区)≤(F2c或F2，以及，R2c或R2)≤(F1c或F1，以及，Rlc或Rl)。
根据本发明所述的合成核酸的方法，作为优选，所得到的第二核酸可以通过引入加速探针Xin的方法使得核酸扩增加速进行，其中Xin是位于F1区到R1区的中间区段。
本发明可以以步骤3)置换后的互补链为模板重复步骤1)-3)合成长链核酸。
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，允许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：
一寡核苷酸I，其包括F1区和R1r区，所述R1r区与F1区的5’侧相连，其中，
F1区：具有与模板的F1c区互补的核苷酸序列的区，和
R1r区：与模板的R1区反向的区；
一寡核苷酸II，其包括R1区和F1r区，所述F1r区与R1区的5’侧相连，其中，
R1区：具有与模板的R1c区互补的核苷酸序列的区，和
F1r：与模板的F1区反向的区；
第一引物I，其具有与作为模板的核酸中F1c区3'侧的F2c区互补的核苷酸序列；
第二引物II，其具有与作为模板的核酸中R1c区3'侧的R2c区互补的核苷酸序列；
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探针Xin，其中所 述Xin是位于模板的F1区到R1区的中间区段的核苷酸片段，具体地，即位于F1至R1之间的Fin及Rin区域。
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螺旋环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。
具有首尾为互补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首位并排连接在单链里的核酸。此外，本发明中应包含用于在互补链间成螺旋的核苷酸序列。本发明中该序列称为成螺旋环序列。本发明合成的核酸基本上由通过成螺旋环序列连接的互相互补的链组成。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，本发明具有互补核苷酸序列首尾连接在单链里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，并且其退火产物，在弯曲部分组成不涉及碱基配对的环。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。
本发明合成的具有首尾可互补成环序列的单链核苷酸与天然存在的核酸不可能有相同的结构，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。而且产物可为DNA亦可为RNA。生成的产物通过结合实现核酸聚合反应的引物结构，聚合反应底物类型，聚合反应的试剂而定。
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F1r区具备在3’-末端与同一链上的部分Flc区退火形成合成互补链。有许多关于互补链合成反应的报道，其中形成发夹环，以发夹环序列自身为模板，亦有形成螺旋环，以螺旋环序列自身为模板，而本发明中提供给螺旋环其首尾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，先前以螺旋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，功能为合成互补链的模板的，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’-末端被提供F1r区，可与同一链上的部分Flc退火，通过该F1r区与同一链上的Flc退火，可形成包含可碱基配对的R1区在内的环，可在各种方法中得到该核酸。
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和从退火为F2c外引物F2为合成起点合成互补链以及同时替换先前合成的互补链，外引物F2位于F1c的3’-侧。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；以与R2c退火的R2作为合成起点的互补链的合成及同时的链置换发应，R2位于R2c的3’-侧。在所给反应条件下不能预计反转录酶显示DNA/RNA链置换活性时，可以结合具有如上述链置换活性的DNA聚合酶。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
这些酶中，Bst DNA聚合酶或Bca(exo-)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。
本发明合成具有首尾序列可退火成环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首尾连接的闭合环。能碱基配对的环的结构在图3中显示。如图3中看到的该环由核苷酸序列F1，R1，F1c，R1c构成，可进行分子内退火形成闭合环。
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螺旋环。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是本发明合成第一核酸的步骤图解。
图2是本发明合成第二核酸的步骤图解。
图3是通过本发明单链核酸所形成的环结构的图解。
图4是通过本发明单链核酸所形成的环结构通过添加额外引物加速图解。
图5是显示MERS-orf1b靶核苷酸序列中组成寡核苷酸的每个核苷酸序列的位置关系。
图6是是显示通过以MERS-orf1b为模板合成本发明单链核酸的方法获得的产物琼脂糖电泳结果的照片。
泳道1：碧云天O0107DNA Ladder
泳道2：1fmol MERS-orf1b dsDNA
图7是显示MERS-orf1b靶核苷酸序列中组成寡核苷酸的每个核苷酸序列的位置关系。
图8是显示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1中得到的。其中，
泳道1：碧云天O0107DNA Ladder；
泳道2：1fmol MERS-orf1b dsDNA；
泳道3：纯化产物的HindIII消化。
图9是显示在引物的作用下，含MERS-orf1b靶核苷酸序列DNA增加的实时荧光曲线。
图10是显示在引物的作用下，含MERS-orf1b靶核苷酸序列DNA增加的实时荧光曲线。
图11是显示在引物的作用下，通过添加加速引物条件下，不同DNA靶浓度扩增体系的荧光强度随反应时间变化的曲线。
图12是本发明的合成核酸的方法的流程示意图。
图13是本发明合成的核酸螺旋示意图。
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1对于MERS-orf1b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MERS-orf1b(来自于GenBank:NM_001012270.1)为模板尝试的。实验中用到四种引物即Mo1bHF，Mo1bHR，Mo1bF2和Mo1bR2。Mo1bF2和Mo1bR2是置换分别以Mo1bHF和Mo1bHR为合成起点获得的第一条核酸的外引物。因为以Mo1bHF(或Mo1bHR)合成后外引物为互补链合成起点的引物。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Mo1bHF(或Mo1bHR)的退火优先发生。
组成每个引物的核苷酸序列如序列表所示，引物的结构特征在下面概括。此外，针对靶核苷酸序列每个区的位置关系在图5中显示。
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表1所示。
表1引物与寡核苷酸信息
Figure PCTCN2017071394-appb-000001
Figure PCTCN2017071394-appb-000002
通过所述引物，合成R1与R1rc、F1c与F1r互补成螺旋环的核酸。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(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 Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
靶:MERS-orf1b dsDNA/SEQ ID NO.5
具体的各核苷酸序列如下：
SEQ ID NO.l：GTACGAAGGGCATTACGCTCTCGTGTTATTTCCAGG；
SEQ ID NO.2：GGACCTTTATTGTGCTCTCGCATTACGGGAAGCATG；
SEQ ID NO.3：TACCCGCAAATGTCCCATA；
SEQ ID NO.4：TGTAGAGGCACATTGGTG；
SEQ ID NO.5：
Figure PCTCN2017071394-appb-000003
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上样缓冲液力加到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6中显示，各泳道分别相对于下面的样品。
1.分子量标记DNA ladder
2. 1fmolM13mpl8dsDNA。
实施例2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1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1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1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7中显示，各泳道分别相对于下面的样品。
1.分子量标记DNA ladder
2. 1fmolM13mpl8dsDNA
3纯化产物的HindIII消化
实施例3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(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)
120μM
引物：
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
靶:MERS-orf1b dsDNA/SEQ ID NO.5
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为75min。荧光强度随反应时间变化的曲线如图8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4应用基于EvaGreen的实时荧光比较不同浓度靶基因扩增
反应溶液组合(25μL)八管
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 Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
靶:MERS-orf1b dsDNA/SEQ ID NO.5
相同的反应液设置8管，仅靶的添加有区别分别为108Copies，107Copies，106Copies，105Copies，104Copies，103Copies，102Copies，纯水(0Copies)。
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为90min。不同靶浓度扩增体系的荧光强度随反应时间变化的曲线如图9所示。此结果说明本发明方法所获得的结果线性关系较好，可应用于目标核酸定量检测。
实施例5应用基于EvaGreen的实时荧光比较不同浓度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。
反应溶液组合(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)
10U AMV反转录酶
1X EvaGreen(Biotum)
引物：
1600nM Mo1bHF/SEQ ID NO.l
1600nM Mo1bHR/SEQ ID NO.2
200nM Mo1bF2/SEQ ID NO.3
200nM Mo1bR2/SEQ ID NO.4
靶:MERS-orf1b ssRNA/SEQ ID NO.5
相同的反应液设置8管，仅靶的添加有区别，分别为108Copies，107Copies，106Copies，105Copies，104Copies，103Copies，102Copies，纯水(0Copies)。
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为90min。不同靶浓度扩增体系的荧光强度随反应时间变化的曲线如图10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Fin对于不同浓度靶基因扩增
反应溶液组合(25μL)八管
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 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
靶:MERS-orf1b dsDNA/SEQ ID NO.5
Fin及Rin引物的具体核苷酸序列如下：
SEQ ID NO.6：ACAGTTCCTGGATATCCTAAGCT；
SEQ ID NO.7：ACAGCCTCTTCACGAGTAATG。
相同的反应液设置8管，仅靶的添加有区别，分别为106Copies，105Copies，104Copies，103Copies，102Copies，101Copies，100Copies，纯水(0Copies)。
设置ABI StepOne real time PCR反应温度恒定为63℃，反应时间为75min。不同靶浓度扩增体系的荧光强度随反应时间变化的曲线如图11所示。此结果同实施例6中的结果对比说明加入Fin寡核苷酸可明显减少扩增时间。
当然，本发明还可以有多种实施例，在不背离本发明精神及其实质的情况下，熟悉本领域的技术人员可根据本发明的公开做出各种相应的改变和变型，但这些相应的改变和变形都应属于本发明所附的权利要求的保护范围。

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3’末端具有与同一条链上的F1c区退火的F1r区，并且通过所述F1r区与Flc区的同时退火形成螺旋环；
    2)以步骤1)所述核酸为模板合成其自身的互补链，所述互补链的R1rc区与R1区退火，而Flc区退火的F1r区的3'末端组成螺旋环，作为合成起点；
    3)通过聚合酶催化链置换型互补链合成反应而进行互补链合成，以置换步骤2)中所合成的互补链，其中所述多核苷酸在其3’末端包含一种与步骤2)中合成的互补链的任意区域互补的序列。
  2. 根据权利要求1所述的合成核酸的方法，其特征在于，步骤1)所述核酸的制备方法，包括以下步骤：
    i)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3’侧的F2c区，该模板的5’末端包括R1区和位于R1区5’侧的R2区，其中所述第一寡核苷酸I，包括R1r区与Fl区，所述Rlr区与F1区的5’侧相连，其中，
    F1区：具有与模板F1c区互补的核苷酸序列的区，
    R1r区：与模板R1区反向的区；
    ii)合成第一核酸的步骤，所述第一核酸具有与所述模板互补的核苷酸序列，此步骤以第一寡核苷酸I的F2区作为合成的起点合成第一核酸，所述第一核酸的3'末端具有与同一条链上的部分F1c区退火的F1区，并且通过所述F1区与Flc区的同时退火形成的螺旋环；
    iii)利用聚合酶催化链置换反应置换步骤ii)中合成的第一核酸，其中与模板中的F1c的3’侧的F2c区退火的第一外引物I用作合成的起点，和
    iv)退火步骤，使第二寡核苷酸II与步骤iii)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F1r区，并且Flr区与R1区的5’侧相连，第二寡核苷酸II为第一寡核苷酸I的反向序列；其中，
    R1区：具有与第一核酸的R1c区互补的核苷酸序列的区，
    Flr区：与第一核酸的F1区反向的区；
    v)以所述第二寡核苷酸II作为合成的起点，合成第二核酸，并利用聚合酶催化链置换反应置换该第二核酸获得步骤1)所述的核酸；其中，置换第二核酸时，与第一核酸中的R1c的3’侧的R2c区退火的第二外引物II用作合成的起点。
  3. 根据权利要求2所述的合成核酸的方法，其特征在于，步骤i)所述模板为RNA，步骤ii)中的第一核酸通过具有反转录酶活性的酶来合成。
  4. 根据权利要求1-3中任一所述的合成核酸的方法，其特征在于，步骤3)合成的核酸是在其一条链上具有首尾互补核苷酸序列的核酸。
  5. 根据权利要求1-3中任一所述的合成核酸的方法，其特征在于，每种寡核苷酸与其在模板中的互补区之间的解链温度存在以下的关系：(外引物或模板 3'侧区)≤(F2c或F2，以及，R2c或R2)≤(F1c或F1，以及，Rlc或Rl)。
  6. 根据权利要求2所述的合成核酸的方法，其特征在于，所得到的第二核酸通过引入加速探针Xin的方法使得核酸扩增加速进行，其中Xin是位于F1区到R1区的中间区段。
  7. 根据权利要求1-3任一所述的合成核酸的方法，其特征在于，以步骤3)置换后的互补链为模板重复步骤1)-3)合成核酸。
  8. 一种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：
    一寡核苷酸I，其包括F1区和R1r区，所述R1r区与F1区的5’侧相连，其中，
    F1区：具有与模板的F1c区互补的核苷酸序列的区，和
    R1r区：与模板的R1区反向的区；
    一寡核苷酸II，其包括R1区和F1r区，所述F1r区与R1区的5’侧相连，其中，
    R1区：具有与模板的R1c区互补的核苷酸序列的区，和
    F1r：与模板的F1区反向的区；
    第一引物I，其具有与作为模板的核酸中F1c区3'侧的F2c区互补的核苷酸序列；
    第二引物II，其具有与作为模板的核酸中R1c区3'侧的R2c区互补的核苷酸序列；
    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。
  9. 根据权利要求10所述的试剂盒，其特征在于，所述试剂盒还包括加速探针Xin，其中所述Xin是位于模板的F1区到R1区的中间区段的核苷酸片段。
  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。
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