CN106701738B - Method for isothermal unwinding of double-stranded DNA and preparation of single-stranded DNA - Google Patents
Method for isothermal unwinding of double-stranded DNA and preparation of single-stranded DNA Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and provides a method for isothermal disentangling of double-stranded DNA and a method for preparing single-stranded DNA based on the method. The technical scheme of isothermal unwinding of the double-stranded DNA is that a recombinase is combined with a single-stranded DNA probe to form a compound, the compound recognizes a sequence in the double-stranded DNA which is homologous with the single-stranded DNA probe and invades the double-stranded DNA, so that isothermal unwinding of the double-stranded DNA is realized, then the single-stranded DNA probe is complementarily paired with one strand of the double strand, and a single-stranded binding protein is combined with the other strand to stabilize an unwinding structure. Based on this scheme and the property that the 3' -end of the single-stranded DNA probe can be recognized by enzymes such as ligase and polymerase, the present invention also proposes a scheme for preparing single-stranded DNA and introduces a scheme for preparing single-stranded DNA by ligase or elongase in detail. The technical scheme can be completed under the isothermal condition, an ATP circulating system is not needed, even ATP can be replaced by dATP, the used enzyme is wide in source and easy to obtain, and a general platform is provided for the isothermal technology by taking double-stranded DNA as a research object.
Description
Technical Field
The invention belongs to the technical field of molecular biology, relates to unwinding of double-stranded DNA and preparation of single-stranded DNA, and particularly relates to a universal method for isothermal unwinding of double-stranded DNA by using recombinase and single-stranded DNA and a method for preparing single-stranded DNA by using the universal method.
Background
DNA is a vector for a gene that carries genetic information encoding the amino acid sequences of all structural and functional proteins in the cell. The genome in organisms is almost present in a double-stranded form except for a small portion of the virus. However, many basic in vitro DNA-based procedures such as DNA hybridization, ligation, amplification, etc. utilize the principle of base-complementary pairing between probes or primers and the target DNA sequence. Therefore, the unwinding of double-stranded DNA is a prerequisite for all basic DNA manipulations and is critical for a series of subsequent applications such as gene detection, sequencing, editing, etc. The common methods for unwinding double strands are classified into physical methods and chemical methods. The physical method is mainly a thermal denaturation method in which two single strands are separated by heating. The chemical method mainly utilizes denaturants such as urea, strong alkali and the like to destroy hydrogen bonds formed between the two single chains. Although both methods completely open the double strand, the former is limited by a heating device, which not only increases the cost and reduces the portability, but the latter is not suitable for many biochemical reactions such as enzymatic reaction because the reaction conditions are too severe and the composition is complicated. Therefore, the establishment of an isothermal, convenient, economic and biocompatible double-stranded DNA unbinding method has important scientific significance and practical value.
Since most of the work only needs to be done on partial sequences in genomic DNA, the extraction of target sequences is a primary task. The commonly used technique is PCR amplification, but this technique requires thermal denaturation and repeated thermal cycling, and is highly instrument-dependent and complicated to operate. With the increasing demand for portability, economy and rapidity of instruments and devices, isothermal reaction-based technologies and devices are preferred. However, most of the isothermal reactions can only be directly studied on single-stranded DNA, such as the very representative isothermal gene detection technology-nucleic acid sequence-dependent amplification technology (NASBA), strand displacement amplification technology (SDA), rolling-over replication technology (LRCA and HRCA), loop-mediated isothermal nucleic acid amplification technology (LAMP), etc. The methods currently used to obtain single strands are mainly: (1) the strategy of combining thermal denaturation, internal and external primers and strand displacement is utilized. Performing thermal denaturation on the double-stranded DNA to open a strand, hybridizing the primer and the template, and replacing an extension product of the inner primer in the extension process of the outer primer to obtain a single strand; (2) linearly accumulating one strand of the double-stranded product by asymmetric PCR; (3) cutting a long genome template into short nucleic acid fragments by endonuclease, and degrading one strand of double strands by using exonuclease such as Lambda, ExoIII and the like to finally obtain the target single strand. Wherein, the methods (1) and (2) can not get rid of thermal denaturation and heating equipment, and the original purpose of isothermal reaction is violated; although the method (3) can avoid the dependence on instruments, the endonuclease needs to recognize specific sequences, so that the universality is greatly reduced. Therefore, it is necessary to design a general scheme capable of preparing single-stranded DNA under isothermal conditions using double-stranded DNA as a template to cooperate with a rapidly developed isothermal technique to establish a system-complete isothermal operation system suitable for a wide range of DNA samples.
Disclosure of Invention
The invention aims to provide a method for unwinding double-stranded DNA under isothermal condition and a method for preparing single-stranded DNA rapidly and simply by using the method without depending on expensive instruments, and provides a universal solution for isothermal reaction technology to directly use double-stranded DNA as a research object.
The technical scheme for realizing the purposes is as follows:
a method for isothermal unwinding of double-stranded DNA, which comprises the following steps: a complementary single-stranded DNA probe is designed for one strand of the target sequence in the double-stranded DNA. In the presence of high concentrations of ATP or dATP, the recombinase first forms a complex with the single-stranded DNA probe, and then double-stranded DNA and single-stranded binding protein SSB are added to the system; the complex scans the double-stranded DNA, which is cleaved when it encounters a region of homology to the single-stranded DNA probe, which pairs with its complementary strand to form a heteroduplex, and the other strand binds to the single-stranded binding protein SSB to form a stable open-stranded structure.
Because the 3 'end of the single-stranded DNA probe used in the technical scheme of isothermal unwinding of the double-stranded DNA can be recognized by a plurality of enzymes, the invention provides the technical scheme for preparing the single-stranded DNA by using the double-stranded DNA as a template, designing the single-stranded DNA probe according to the technical scheme of isothermal unwinding of the double-stranded DNA, unwinding the double strand, and combining the specific enzyme by utilizing the characteristic of the 3' -end of the single-stranded DNA probe: (1) when the used enzyme is ligase, two single-stranded DNA probes can be designed to be continuously complementary with one strand (template strand) of a target sequence in the double-stranded DNA and the 5' -end of the downstream single-stranded DNA probe is phosphorylated, then the two single-stranded DNA probes are continuously complementary with the template strand after double strands are untied and a gap is formed in the middle, and the gap is connected by the ligase to obtain a complete single strand; (2) when the used enzyme is polymerase, two single-stranded DNA probes with opposite directions are designed by respectively using two strands of the double-stranded DNA as templates, after the double strands are untied, the two single-stranded DNA probes are respectively complementary with one strand of the double-stranded DNA, are recognized and extended by the polymerase with strand displacement activity, and simultaneously displace the other strand of the double strands respectively, so that the preparation of the target single-stranded DNA is realized, and more single strands can be obtained by the cyclic reciprocation of the process.
The enzymes used in the technical scheme of the invention are mesophilic enzymes, and can work under isothermal conditions in the whole process without depending on instrument temperature control.
The technical scheme of the invention utilizes high-concentration ATP or dATP without a regeneration system.
The homologous region of the single-stranded DNA probe and the double-stranded template related by the invention is not less than 24 nucleotides, and the single-stranded DNA probe and the double-stranded template can be completely complementarily paired or partially paired.
When the single-stranded DNA probe of the present invention is used in ligation reaction, the 5 'end of the downstream probe must be perfectly complementarily paired and phosphorylated, and the 3' end of the upstream probe must be perfectly complementarily paired. If the single-stranded DNA probe involved is a padlock probe (padlock probe), it is only necessary to ensure that the 5 ' end and the 3 ' end of the probe form a gap after complete complementary pairing with the template and the 5 ' end is phosphorylated.
When the single-stranded DNA probe of the present invention is used for an extension reaction, the 3' end must be perfectly complementary to the template.
The recombinase protein and the single-chain binding protein are both derived from escherichia coli.
The polymerase involved in the invention is mesophilic polymerase, has activity at 25-65 ℃, does not have 5 '-3' exonuclease activity and has strand displacement activity.
As used herein, the following words/terms have the following meanings, unless otherwise specified.
"DNA": deoxyribonucleic acid. Is a biological macromolecule with genetic information, is formed by connecting 4 main deoxyribonucleotides through 3 ', 5' -phosphodiester bonds, and is a carrier of the genetic information.
"PCR": polymerase chain reaction. The method is a method for synthesizing specific DNA fragments in vitro by enzyme, and is carried out in a cycle consisting of a plurality of steps of reaction such as high-temperature denaturation, low-temperature annealing, suitable temperature extension and the like, so that the target DNA can be rapidly amplified, and the method has the characteristics of strong specificity, high sensitivity, simple and convenient operation, time saving and the like.
"sequence of interest": the analytes to be detected include DNA and RNA sequences.
The "complex": the recombinase forms a complex with the single-stranded DNA probe in a form similar to that of the double-stranded DNA, and the recombinase winds around the single-stranded DNA probe and extends spirally.
"mesophilic" or "mesophilic protein": relative to a thermophilic enzyme such as Taq DNA polymerase. Here, mesophilic enzyme means an enzyme which does not resist high temperature within a working temperature range of 15 to 70 ℃ such as Bsm DNA polymerase (Thermo Fisher. TM., 30 to 63 ℃), Bst DNA polymerase (NEB, <70 ℃), T4DNA ligase (NEB, recommended reaction temperature of 16 ℃, 20 to 25 ℃), T4 polynucleotide kinase (NEB, recommended optimum reaction temperature of 37 ℃) and the like.
"isothermal" or "isothermal conditions": the working temperature of the mesophilic enzyme used may refer to a constant temperature condition in the working temperature range of the mesophilic enzyme, or may refer to a temperature condition that dynamically changes in the working temperature range.
The key point of the method disclosed by the invention is that the purpose of separating double-stranded DNA is achieved by utilizing the synergistic effect of recombinase protein, a single-stranded DNA probe and single-stranded binding protein; after forming an open chain structure, the 3' end of the single-stranded DNA probe can be recognized by ligase and polymerase, and single-stranded DNA is prepared through connection and extension reaction; so that the isothermal reaction technology which can only use single strand as the initial template at present can also use double strand as the research object. The invention has obvious advantages over the prior art, and the main advantages thereof comprise:
1. is novel. The invention successfully establishes the method for isothermal double strand unwinding in vitro based on the in vivo recombinant enzyme system, only needs high-concentration ATP or dATP without a regeneration system; the single-strand preparation strategy established on the basis of isothermal unwinding of double strands is novel and simple, and is pioneered at home and abroad.
2. And (4) universality. The invention relates to a scheme for untwisting double-stranded DNA and preparing single strands by using the same, aiming at different purposes, the purpose can be realized only by reasonably designing the complementary number and the position of basic groups of a single-stranded DNA probe, and the scheme is a universal method.
3. And (5) practicability. All reactions can be carried out under isothermal conditions, complex and violent treatments such as heating or chemical denaturation are not needed, the method is suitable for almost all isothermal reactions taking double-stranded DNA as an object, and the application range of the existing isothermal reactions only taking single-stranded DNA as the object is remarkably expanded.
4. And (4) economy. The key proteins involved in the invention are all derived from escherichia coli, and have wide sources and are easy to obtain.
Drawings
FIG. 1 is a schematic diagram showing the process of isothermal unwinding of double-stranded DNA based on a single-stranded DNA probe and a recombinase in example 1.
FIG. 2 is a schematic flow chart of the ligation reaction between single-stranded DNAs by isothermal unwinding of double-stranded templates in accordance with specific example 2.
FIG. 3 is a schematic flow chart of an extension reaction of a single-stranded DNA by unwinding a double-stranded template in specific example 3.
FIG. 4 is a schematic flow chart of the specific example 4 for solving the plasmid to provide a template for helicase-dependent isothermal amplification reaction.
FIG. 5 is a graph showing the results of example 1.
FIG. 6 is a graph showing the results of example 2.
FIG. 7 is a graph showing the results of example 3.
FIG. 8 is a graph showing the results of example 4.
Detailed Description
The invention will be further illustrated by way of example with reference to the accompanying drawings. It will be understood by those skilled in the art that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 isothermal Unlysis of double-stranded DNA based on Single-stranded DNA probes and recombinase
In order to verify the mechanism of isothermal unwinding of double-stranded DNA based on a single-stranded DNA probe and a recombinase by radioisotope labeling and polyacrylamide gel electrophoresis, the single-stranded DNA probe is designed to be as long as the double-stranded DNA, according to the technical scheme of isothermal unwinding of double-stranded DNA of the present invention, the single-stranded DNA probe can directly and completely open the double-stranded DNA and the complementary strand thereof for pairing and replacing with another non-complementary strand, without requiring a stable open-stranded structure of single-stranded binding protein (please refer to examples 2-4 for studies related to stable open-stranded structures of single-stranded binding protein, wherein the single-stranded DNA probe is far shorter than the double-stranded DNA template). The steps for isothermal unwinding of double stranded DNA templates are shown in FIG. 1. Design of Single-stranded DNA Probe X1 with isotope P at its 5' end32And (4) marking. In the presence of ATP or dATP, the recombinase, RecA, forms a complex with X1, and cleaves the double-stranded DNA template ds (formed by annealing ds1 and ds 2), X1, andone of the complementary strands forms a pair and is displaced from the other non-complementary strand, thereby obtaining P32A labeled double strand and one unlabeled single strand.
(1) Single-stranded DNA Probe X1, double-stranded template ds sequence
X1:5’-TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTA GGAGCT AAA-3’
ds1:5’-TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTAGGA GCT AAA-3’
ds2:5’-TTT AGC TCC T AG ACC TTT AGC AGC AAG GTC CAT ATC TGA CTT TTTGTT AAC GTA-3’
(2) Reaction System and conditions
(3) Results of the reaction
As shown in FIG. 5, the reaction with the addition of RecA resulted in a labeled double strand on polyacrylamide gel electrophoresis with only a small background band without the addition of RecA.
Example 2A double-stranded template T12 (formed by annealing T1, T2) was cleaved with a group A single-stranded DNA probe (X1, X1f), and then ligated with a ligase to prepare a single-stranded DNA by ligation.
The procedure for preparing single-stranded DNA by ligation reaction by unbinding the double-stranded template is shown in FIG. 2. Two single-stranded DNA probes (X1, X1f) were designed, wherein the 5 ' -end of X1f was modified by phosphorylation, the 3 ' -end of X1 was hydroxyl, and the 5 ' -end was labeled with isotope P32And (4) marking.
In the presence of ATP or dATP, the recombinase RecA forms a complex with the single-stranded DNA probes X1, X1f, respectively, which scans the double-stranded T12. When the regions homologous to X1 and X1f are scanned, the double strand T12 is cleaved, X1 and X1f are paired with the complementary strands respectively to form a gap in the middle, X1 is at the 5 'end and X1f is at the 3' end. The other displaced strand binds to the single-stranded binding protein SSB, preventing its re-pairing from causing the two invaded single-stranded DNA probes to fall off, thereby stabilizing the formed open-stranded structure.
The ligase condenses the 3 'hydroxyl of X1 with the 5' phosphate of X1f in the presence of ATP or dATP to form a phosphodiester bond, thereby covalently joining X1 to X1f to form a single intact chain.
(1) Single-stranded DNA probe X1, X1f, double-stranded template T12 sequence
X1:5’-TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTA GGAGCT AAA-3’
X1f:5’-TCA TCT TGT AGT CCA TTG TAA TTG TAA ATA GTA ATT GTC CCT ATAGTG AGT CGT-3’
T1:GAA TTC TAA TAC GAC TCA CTA TA GGG ACA ATT ACT ATT TAC AAT TAC AATGGA CTA CAA GA TGA TTT AGC TCC TAG ACC TTT AGC AGC AAG GTC CAT ATC TGA CTTTTT GTT AAC GTA
T2:TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTA GGAGCT AAA TCA TCT TGT AGT CCA TTG TAA TTG TAA ATA GTA ATT GTC CCT ATA GTG AGTCGT ATT AGA ATT C
(2) Reaction System and conditions
The reaction conditions are as follows: the reaction system was mixed with the components except SSB, T12, and T4DNAligase, incubated at 37 ℃ for 5 minutes, and then the remaining 3 components were added and incubation continued for 1 hour.
The 2 negative controls were: systems that do not contain RecA and SSB and systems that do not contain a double-stranded template.
(3) Results of the reaction
As shown in FIG. 6, isotopically labeled X1 is linked to X1f to form a longer chain and thus has a reduced mobility in denaturing polyacrylamide gel electrophoresis. While the systems without RecA and SSB and without double-stranded template did not yield the corresponding ligation products.
Example 3A double-stranded template T12 (formed by annealing T1, T2) was cleaved with a group B single-stranded DNA probe (X1, X1r), and a single-stranded DNA was prepared by extension reaction with polymerase.
The procedure for preparing single-stranded DNA by extension reaction by unwinding the double-stranded template is shown in FIG. 3. Design two Single-stranded DNA probes (X1, X1r), X1 with isotope P at the 5' end32And the label is complementarily paired with the 3' downstream region of the T1 strand in the double-stranded template. X1r is complementarily paired with the 3' downstream region of the T2 strand in the double-stranded template.
In the presence of ATP or dATP, the recombinase RecA forms a complex with the single-stranded DNA probes X1, X1r, respectively, which scans the double-stranded T12. When the sequences homologous with X1 and X1r are scanned, the double-stranded T12 is opened in the corresponding regions, X1 and X1r are respectively paired with the complementary strands thereof, and the strand of the two regions, of which the other strand is replaced, is combined with the single-stranded binding protein SSB, so that the phenomenon that the two invaded single-stranded DNAs are separated due to the re-pairing is prevented, and the formed open-stranded structure is stabilized.
The polymerase having strand displacement activity introduces dNTPs one by one at the 3' ends of X1 and X1r in the presence of a mixture of dNTPs to extend the single-stranded DNA probe while the other strand is completely displaced during the extension to obtain a single-stranded DNA. The double-stranded DNA formed by the probe extension product and the complementary strand thereof can be used as a new template to start a new round of reaction processes of strand opening, extension and replacement, and the whole process is circularly carried out, so that more single-stranded DNA probes are extended and single-stranded DNA is obtained. (1) Single-stranded DNA probe X1, X1r, double-stranded template T12 sequence
X1:5’-TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTA GGAGCT AAA-3’
X1r:5’-GAA TTC TAA TAC GAC TCA CTA TA GGG ACA ATT ACT ATT TAC AAT TACAAT GGA C-3’
T1:GAA TTC TAA TAC GAC TCA CTA TA GGG ACA ATT ACT ATT TAC AAT TAC AATGGA CTA CAA GA TGA TTT AGC TCC TAG ACC TTT AGC AGC AAG GTC CAT ATC TGA CTTTTT GTT AAC GTA
T2:TAC GTT AAC AAA AAG TCA GAT ATG GAC CTT GCT GCT AAA GGT CTA GGAGCT AAA TCA TCT TGT AGT CCA TTG TAA TTG TAA ATA GTA ATT GTC CCT ATA GTG AGTCGT ATT AGA ATT C
(2) Reaction System and conditions
The reaction conditions are as follows: firstly, except SS in the reaction systemB. T12, dNTPs and other components of Bsm DNA polymerase were mixed and incubated at 37 ℃ for 5 minutes, followed by addition of the remaining 4 components and addition of MgCl to a final concentration of 6mM and incubation continued for 1 hour.
The 2 negative controls were: systems that do not contain RecA and SSB and systems that do not contain a double-stranded template.
(3) Results of the reaction
As shown in FIG. 7, the single strand displaced during the X1 extension process can be used as the extension template of X1r, and the single strand displaced during the X1r extension process can also be used as the extension template of X1, so that many single strand templates can be generated during multiple rounds of extension processes. Isotopically labeled X1 is extended by polymerase and thus has reduced mobility in denaturing polyacrylamide gel electrophoresis. While the systems without RecA and SSB and without double-stranded template did not yield the corresponding extension products.
Example 4 plasmid T1-ds (obtained by cloning T12 into the vector T1) was cleaved with group B single-stranded DNA probes (X1, X1r) to provide a template for helicase-dependent isothermal amplification reactions.
The reaction steps for unwinding the plasmid to provide a template for the helicase-dependent isothermal amplification reaction are shown in FIG. 4.
In the presence of ATP or dATP, recombinase RecA forms a complex with single-stranded DNA probes X1 and X1r respectively, after the complex is scanned on a plasmid and is homologous with X1 and X1r, the plasmid is untied in corresponding regions, X1 and X1r are respectively paired with complementary strands of the plasmid, and the strand which is replaced from the other two regions is combined with single-stranded binding protein SSB to prevent the re-pairing of the two regions from causing the shedding of two invaded single-stranded DNAs. The polymerase having strand displacement activity introduces dNTPs one by one at the 3' ends of X1 and X1r in the presence of a mixture of dNTPs to allow the oligonucleotide to be extended while the other strand is completely displaced during extension. The other strand is replaced to be used as a template of X1 and X1r to perform extension reaction, and two rounds of circulation can be carried out to obtain a blunt-ended double strand. The double chain with the blunt end can be separated into two single chains by helicase, the primers P-X1 and P-X1r can be respectively and complementarily paired with the two single chains and extended under the action of polymerase, and the cycle is repeated, so that the helicase-dependent isothermal amplification reaction can be completely carried out under the isothermal condition and the final amplification product is produced.
(1) Single-stranded DNA probe X1, X1r, primer P-X1, P-X1r sequence
X1:5’-TACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTAGGAGCTAAA-3’
X1r:5’-GAA TTC TAA TAC GAC TCA CTA TA GGG ACA ATT ACT ATT TAC AAT TACAAT GGA C-3’
P-X1:5’-TAGTAGAGTCCTGA TAC GTT AAC AAA-3’
P-X1r:5’-CTACAGAGTCAGTC GAATTCTAAT AC-3’
(2) Reaction System and conditions
Plasmid isothermal melting reaction system (4 ul):
directly adding the mixed plasmid isothermal melting system as a template into a helicase-dependent isothermal amplification reaction system, wherein the steps are as follows:
the whole reaction was incubated at 37 ℃ for 1 hour
The 2 negative controls were: the helicase dependent isothermal amplification system directly takes a 62.5pM plasmid as a template, and the plasmid is not treated by a plasmid isothermal open-chain system; the helicase-dependent isothermal amplification system does not add any template.
(3) Results of the reaction
As shown in FIG. 8, the obvious amplification band is obtained by adding the helicase-dependent isothermal amplification system after the plasmid is mixed with the isothermal double strand unwinding system in advance, while the obvious amplification band is not obtained by adding the plasmid directly or without adding any template in the helicase-dependent isothermal amplification system.
SEQUENCE LISTING
<110> institute of biological research of Chengdu of Chinese academy of sciences
<120> method for isothermal disentangling of double-stranded DNA and preparation of single-stranded DNA
<130>2016
<160>9
<170>PatentIn version 3.3
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ctacagagtc agtcgaattc taatac 26
Claims (6)
1. A method for isothermal unwinding of double-stranded DNA, comprising: realizing the melting of the double-stranded DNA under the isothermal condition by using a single-stranded DNA probe, a recombinase, a single-stranded binding protein and dATP; the single-stranded DNA probe is designed by taking double-stranded DNA as a template; in the presence of dATP, the recombinase and the single-stranded DNA probe form a complex, the complex recognizes a sequence in the double-stranded DNA which is homologous to the single-stranded DNA probe and invades the double-stranded DNA, so that isothermal open strand of the double-stranded DNA is realized, then the single-stranded DNA probe is complementary to one strand of the double-stranded DNA, and the single-stranded binding protein is bound to the other strand of the double-stranded DNA to stabilize an open strand structure; wherein the working concentration of dATP was 5 mM.
2. The method for isothermal unbinding of double stranded DNA according to claim 1, wherein: the recombinase and the single-chain binding protein are mesophilic proteins and can work under isothermal conditions.
3. The method for isothermal unbinding of double stranded DNA according to claim 1, wherein: the length of the single-stranded DNA probe is not less than 24 bases.
4. The method for isothermal unwinding of double-stranded DNA according to claim 1, wherein: the 3' -end of the single-stranded DNA probe can be recognized by an enzyme and reacted.
5. A method for preparing single-stranded DNA, characterized in that: comprising preparing a single-stranded DNA by cleaving a double-stranded DNA by the method of claim 1 and utilizing the property that the 3' -end of the single-stranded DNA probe can be recognized by a ligase to cause a reaction; specifically, a double-stranded DNA template is prepared, one strand of the double-stranded DNA is taken as the template to design two single-stranded DNA probes which are continuously complementary with the template, the 5' -end of the single-stranded DNA probe positioned at the downstream is phosphorylated, the probe can be continuously complementary with the template after the double strand is opened, and the two single-stranded DNA probes can be connected into a complete single-stranded DNA under the action of ligase.
6. The method for preparing single-stranded DNA according to claim 5, wherein: the enzyme is mesophilic and can work under isothermal condition.
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Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004081224A2 (en) * | 2003-03-11 | 2004-09-23 | Gene Check, Inc. | Reca-assisted allele specific oligonucleotide extension method |
WO2007032837A2 (en) * | 2005-08-11 | 2007-03-22 | The J. Craig Venter Institute | Method for in vitro recombination |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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Non-Patent Citations (1)
Title |
---|
Kinetics of the ATP and dATP-mediated formation of a functionally-active RecA-ssDNA complex;Sunil Nayak等;《Biochemical and Biophysical Research Communications》;20150620;第463卷;第1257-1261页 * |
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