WO2022261874A1 - 一种通过单链滚环扩增获得双链序列的方法 - Google Patents

一种通过单链滚环扩增获得双链序列的方法 Download PDF

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WO2022261874A1
WO2022261874A1 PCT/CN2021/100436 CN2021100436W WO2022261874A1 WO 2022261874 A1 WO2022261874 A1 WO 2022261874A1 CN 2021100436 W CN2021100436 W CN 2021100436W WO 2022261874 A1 WO2022261874 A1 WO 2022261874A1
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stranded
dna
circular dna
sequence
reaction
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PCT/CN2021/100436
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French (fr)
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王冀
谭涛
王欧
章文蔚
陈奥
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深圳华大生命科学研究院
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Priority to CN202180099520.0A priority Critical patent/CN117529561A/zh
Priority to PCT/CN2021/100436 priority patent/WO2022261874A1/zh
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis

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  • the invention relates to the field of biotechnology, more specifically, to a method for obtaining double-stranded sequences by single-stranded rolling circle amplification.
  • Rolling Circle Amplification (RCA) or Rolling Circle Replication (Rolling Circle Replication, RCR) reactions usually refer to the use of DNA polymerases with strand displacement activity (such as Phi29 polymerase, Bst polymerase, Bsu polymerase, Klenow Fragment, Vent polymerase, Pol III polymerase, etc.), polymerase chain reaction using single-stranded circular DNA molecules as templates.
  • DNA polymerases with strand displacement activity such as Phi29 polymerase, Bst polymerase, Bsu polymerase, Klenow Fragment, Vent polymerase, Pol III polymerase, etc.
  • DNA polymerase with strand displacement activity will use the single-stranded circular DNA as a template to The added DNA or RNA fragments are used as primers to amplify the template DNA.
  • this DNA polymerase does not have 5' ⁇ 3' exonuclease activity, when the amplification completes a cycle, the polymerase will unwind one of the DNA double strands in the forward direction and continue the reaction, thereby obtaining a DNA containing multiple identical copies of single-stranded DNA molecules.
  • the polymerase will unwind one of the DNA double strands in the forward direction and continue the reaction, thereby obtaining a DNA containing multiple identical copies of single-stranded DNA molecules.
  • a break or base deletion in one strand of a circular double-stranded DNA molecule such as a plasmid, a viral DNA molecule, etc.
  • the rolling circle amplification reaction involving a polymerase with strand displacement activity is often called rolling circle isothermal amplification reaction due to its advantages of fast reaction speed, high fidelity and constant reaction temperature, and has been widely used.
  • non-circular DNA molecules can also undergo isothermal amplification reactions based on the above principles.
  • the reaction time of rolling circle amplification is long enough, the generated DNA single strands will be folded and entangled with each other, forming a complex spatial secondary structure, and coordinating with metal ions to form a dense DNA molecular nanoball (DNA Nanoball, DNB for short).
  • DNP DNA Nanoparticle
  • DNF DNA Nanoflower
  • DNB has many properties that conventional DNA molecules do not have, such as resistance to DNase digestion, colloid formation with metal ions, and high cell affinity.
  • DNB has the above advantages, its main composition is single-stranded DNA molecules. Compared with double-stranded DNA molecules, RCA products still have great limitations in terms of stability and the diversity of enzyme reactions. Therefore, converting single-stranded RCA products into double-stranded products will greatly expand the application of RCA, and can also form complementary effects through joint application with other technologies to promote technological upgrading.
  • Phi29 and its mutants are currently widely used amplification enzymes in the RCA reaction.
  • Phi29 has a strong strand displacement activity, and has a certain template jump (Template Switch) reactivity, that is, during the reaction, when Phi29 is at the position of the replication fork, especially in the initial stage of the reaction, it has a certain probability It will be replaced on the nearby single-stranded DNA, and use this DNA as a template to replicate in the direction opposite to the original direction of RCA, and finally form a double-stranded DNA product.
  • the RCA product is a double-stranded product, it is a by-product of the RCA reaction and generally has a low yield.
  • the side reaction often occurs in the initial stage of the reaction. Therefore, the resulting double-stranded RCA product has a short length and a small amplification factor, and the product cannot be effectively utilized.
  • Double-primer RCA reaction double-primer RCA, that is, add a reverse primer complementary to the DNA single-stranded loop and a forward primer consistent with the template sequence in the RCA reaction, and perform the RCA reaction at the same time.
  • the reverse primer that is complementary to the template sequence will first bind to the DNA single-stranded loop for RCA reaction, and then the forward primer that is consistent with the template sequence will bind to the generated RCA product, using the RCA product as a template, in the Under the action of Phi29, the extension continues to form a double-stranded RCA product. This process is called the second-strand synthesis of DNB.
  • the double-primer RCA reaction is the most commonly used isothermal amplification system.
  • the advantage of this system is that it can generate a large number of double-stranded DNA copies in a short period of time and obtain relatively stable amplification products.
  • the disadvantage of this method is also obvious. Because the amount of each primer added is difficult to control accurately, the RCA reaction will continue to be repeated in different branch chains.
  • Random primer RCA reaction (R2C2, Rolling Circle Amplification to Concatemeric Consensus Method): The random primer method is similar to the double primer method, the difference is that the random primer can be combined at any position of the single-stranded RCA product, and the double-stranded RCA obtained by the random primer method The product will be more complex, and the second-strand synthesis is more complete than the double-primer RCA method. Exonuclease is needed in the reaction to digest the excess single-stranded DNA generated to ensure as many double-stranded DNA as possible. Although this method can obtain more complete double-stranded RCA products, but because there are too many sites for random primer binding, many branched chain structures will be formed, which is still somewhat different from conventional chain-like DNA molecules, and is not complete in nature. unanimous.
  • Loop-mediated isothermal amplification (LAMP) RCR reaction add adapters with complementary paired sequences to both ends of the single-stranded DNA molecule to form a stem-loop structure. After one-step isothermal amplification, the single strand is converted into a double-stranded structure, and another primer complementary to the stem-loop structure is added for extension to open the double-stranded structure. The opened single-stranded 3' end adapter will undergo intramolecular folding to form a stem-loop structure again, and a double-stranded DNA molecule with two copies can be obtained through one-step isothermal amplification. This process is repeated continuously to obtain a long double-stranded DNA molecule with a repeating sequence.
  • LAMP Loop-mediated isothermal amplification
  • the LAMP method is the main method to obtain a large number of multi-copy long fragment molecules through isothermal amplification.
  • the method has the advantages of fast speed, complete double-strand conversion, and less by-products. However, because more adapters, tag sequences, etc. need to be added during the reaction, there will be more redundant information in the final generated molecule.
  • Hairpin RPA Amplification It is a method proposed by Shanghai Haoqin Biotechnology Co., Ltd. that can self-assemble short molecules to form long polymer molecules. It is similar to LAMP but the implementation method is simpler. The method is to connect the linker with (...TATATATA%) palindromic sequence to both ends of the target fragment, because the annealing temperature of the TA sequence is low, and the linker part will melt when the temperature gradually rises. Under appropriate circumstances, the characteristic of intramolecular pairing (namely, DNA breathing) will occur, and the folded and paired 3' end will serve as a new extension site, which will be extended intramolecularly under the action of the amplification enzyme, so that the original molecule Double the length.
  • the adapter design of the hairpin adapter amplification method is simpler.
  • the disadvantage is that the temperature control of DNA respiration needs to be adjusted, and the TA palindromic sequence will produce non-specific amplification products.
  • the above-mentioned prior art has the following disadvantages: 1) incomplete synthesis of the two strands: in the existing multiple RCA schemes, there is incomplete synthesis of the two strands, and most of the obtained double-stranded RCA products have some single-chain Gap 2) Complex secondary structure: In the above-mentioned several main two-strand synthesis methods, because the amount of primers added cannot be accurately controlled, and all require a long reaction time, the double-strand RCA products generated often have complex secondary structures.
  • the present invention provides a simple solution for obtaining double-chain RCA products.
  • the present invention provides a method for obtaining a double-stranded sequence by single-stranded rolling circle amplification, the method comprising:
  • the single-stranded circular DNA is obtained by circularizing a DNA sample or a cDNA sample, and introducing special bases or specific sequences into the single-stranded circular DNA by PCR or linker ligation.
  • the first primer is a DNA primer or an RNA primer.
  • the breaking mechanism that can cause the opening of the single-stranded circular DNA is that the single-stranded circular DNA contains a specific region that is broken by a biochemical reaction, thereby causing the single-stranded Circular DNA opens.
  • the specific region includes specific bases and/or specific sequences.
  • the special base is inosine deoxynucleotide (I), deoxyuridine nucleotide (dU), RNA base, AP site or methylation site.
  • the specific sequence is a restriction enzyme recognition site or a specific binding site for a protein.
  • the restriction endonuclease recognition site is a region rich in AT base sequence; preferably, the restriction endonuclease recognition site is a Chlamydomonas endonuclease recognition site or Neurospora crassa endonuclease recognition site.
  • the specific binding site of the protein is the recognition region of the guide RNA in the CRISPR/Cas gene editing system; preferably, it is the recognition region of the guide RNA in the CRISPR/Cas9.
  • the single-stranded DNA binding protein, pyrophosphatase and TE buffer are added during or after the rolling circle amplification reaction of the single-stranded circular DNA.
  • the helicase is added during or after the rolling circle amplification reaction on single-stranded circular DNA.
  • the helicase is a type A helicase capable of unwinding in the 3' to 5' direction, preferably Rep helicase, UvrD helicase, Heli308 helicase, PcrA helicase , or RecD2 helicase.
  • the present invention provides a method for constructing a nucleic acid sequencing library, the method comprising:
  • the nucleic acid sequencing library is constructed using LFR technology (Long Fragment Reading Technology) to obtain the nucleic acid sequencing library.
  • the nucleic acid sequencing library is a full-length mRNA transcript library.
  • the present invention provides a sequencing method, the method comprising:
  • the sequencing is high throughput sequencing.
  • the high-throughput sequencing is second-generation sequencing or third-generation sequencing.
  • the method of the present invention has the following advantages: the amplification reaction does not need to add exogenous primers, and the by-products are less; the generated double-stranded RCA product is single and unbranched; the generated double-stranded RCA product is easy to capture and purify; the reaction is a single tube
  • the reaction and operation are simple and easy; the helicase assists in reducing the complexity of the secondary structure, and the product is closer to the conventional DNA molecular structure; the generated complete long-fragment double-stranded multi-copy DNA can be applied to single-molecule sequencing, especially the ONT platform, multiple Copying DNA molecules helps to improve the accuracy of single-molecule sequencing.
  • Figure 1 shows a schematic diagram of single-stranded circular DNA with a special base or specific sequence (A) and a schematic diagram of conventional RCA reaction products (B).
  • Fig. 2 schematically shows the method for obtaining double-stranded sequences by single-stranded rolling circle amplification of the present invention.
  • Figure 3 shows a schematic diagram of the principle of combining the helicase in the 3'-5' direction with the rolling circle amplification sequence.
  • Figure 4 shows the effect of helicase on the efficiency of second-strand synthesis.
  • Figure 5 shows the results of agarose gel electrophoresis of RCA products with complex secondary structures.
  • Figure 6 shows the coverage length versus coverage ratio for Sample 1 assembly.
  • Figure 7 shows the relationship between sample 1 transcript length and assembly coverage.
  • Figure 8 shows the coverage length distribution of Sample 1 assembly.
  • the present invention proposes a simple and easy method for obtaining double-stranded RCA products.
  • a single-stranded circular DNA is subjected to a rolling circle amplification reaction with a first primer to obtain an amplified sequence, and then the amplified sequence is obtained through a breaking mechanism (such as the single-stranded circular DNA). special bases or specific sequences on the DNA) to open the single-stranded circular DNA template to obtain a single-stranded linear DNA; finally, use the single-stranded linear DNA as a primer to perform a reverse RCA reaction to obtain Amplified double-stranded RCA product.
  • the method of the present invention may include four steps: the first step includes preparing a single-stranded circular DNA with a special base (or specific sequence) capable of opening the single-stranded circular DNA;
  • the second step includes performing an RCA reaction on the single-stranded circular DNA with the first primer to obtain an amplified sequence, and a helicase can be added during the RCA reaction or after the RCA reaction to make it participate in the RCA reaction;
  • the third step includes Through a biochemical reaction, the single-stranded circular DNA is broken at a specific base or specific sequence position to form a single-stranded linear DNA;
  • the fourth step includes reverse RCA reaction using the single-stranded linear DNA as a primer , to obtain an amplified double-stranded RCA product.
  • Figure 2 schematically shows the method of the present invention for obtaining a double-stranded sequence by single-stranded rolling circle amplification, and the following sub-steps exemplarily describe the principle of the method of the present invention:
  • the first step prepare single-stranded circular DNA with special bases (or specific sequences).
  • DNA samples can be directly circularized, or cDNA can be obtained by reverse transcription of total RNA and then circularized to prepare single-stranded circular DNA.
  • Special bases or specific sequences can be introduced into single-stranded circular DNA by methods such as PCR and linker ligation, so that when necessary, through biochemical reactions, the single-stranded circular DNA is at the position of special bases or specific sequences Fragmentation occurs, which in turn leads to the opening of the single-stranded circular DNA, resulting in a single-stranded linear DNA strand.
  • A shows a schematic diagram of a single-stranded circular DNA with a specific base or specific sequence.
  • the special bases include but are not limited to I bases (inosine nucleotides), dU bases (uracil deoxynucleotides), RNA bases, AP sites, methylation sites, etc.; Introduce specific sequences, including but not limited to restriction endonuclease recognition sites, etc.
  • I base and dU base are taken as examples to realize the ring-opening reaction, and other ring-opening methods are not listed here one by one.
  • the opening of the single-stranded circular DNA by the special base or specific sequence is preferably controlled, for example, the opening of the single-stranded circular DNA is realized under conditions suitable for biochemical reactions.
  • endonuclease V can be used to digest and excise, so that the single-stranded loop is opened;
  • the dU base can be recognized and excised by UDG enzyme or APE1 enzyme, thereby opening the single-stranded loop;
  • RNA bases can be recognized and excised by RNaseA, RNaseH and other enzymes, thereby opening the single-stranded loop;
  • the AP site can be recognized and excised by the enzyme APE1, thereby opening the single-stranded loop;
  • the methylated C base site can be treated with sodium bisulfite or treated with APOBEC deaminase or TET2 enzyme to convert the methylated C base into a dU base, and then pass UDG enzyme or APE1 The enzyme recognizes and excises, allowing the single-stranded loop to open;
  • Chlamydomonas endonuclease or Neurospora crassa endonuclease can be used to recognize and excise, so that the single-stranded loop is opened;
  • the specific sequence is the recognition sequence of the guide RNA in the CRISPR/Cas gene editing system
  • the mutation-containing CRISPR/Cas9 system on the DNA molecule
  • a single-strand gap SSB Single-Stranded Break
  • any bases and specific sequences that can open the single-stranded DNA can be used as an alternative to this scheme to prepare for the subsequent DNA ring-opening reaction.
  • the present invention is intended to cover any base and specific sequence that can open single-stranded DNA.
  • the second step carry out the RCA reaction.
  • FIG. 1 B shows a schematic diagram of conventional RCA reaction products.
  • components such as single-stranded DNA binding protein, pyrophosphatase and TE buffer to the RCA reaction to obtain a relatively "fluffy" RCA product.
  • the resulting RCA product will still eventually form a compact DNB morphological molecule, which is not conducive to the second-strand synthesis.
  • the two-strand primer input after the RCA reaction is difficult to completely bind to the RCA product; on the other hand, due to factors such as steric hindrance, the two-strand synthetase cannot convert the RCA product into a complete double-strand product.
  • FIG. 3 shows a schematic diagram of the principle of combining the helicase in the 3'-5' direction with the rolling circle amplification sequence.
  • the helicase may be added during the RCA reaction or after the RCA reaction.
  • the helicase utilizes the energy released by the hydrolysis of ATP to undergo a conformational change and at the same time be able to open the hydrogen bond structure of double-stranded DNA.
  • the helicase will specifically bind to the single-stranded DNA, and start the unwinding reaction from 3' to 5' under the action of ATP. Adding helicase during the RCA reaction or after the RCA reaction can help the RCA product to maintain a relatively simple single-chain form, creating conditions for the subsequent reverse RCA reaction.
  • Helicase has directionality, and the present invention adopts the single-stranded DNA helicase (being A-type ⁇ -helicase series; Type A ⁇ -Helicase Family) of 3' to 5' direction as an example to illustrate the principle of the present invention, as Figure 3 shows.
  • the invention can employ the Tte UvrD helicase of NEB.
  • any A-type helicase with the ability to unwind in the 3' to 5' direction such as Rep, Heli308, PcrA, RecD2, etc., can be used as an alternative in this embodiment, and the present invention intends to include these unwinding enzyme.
  • the third step opening the single-stranded circular DNA.
  • the single-stranded circular DNA is broken at specific bases and specific sequence positions through biochemical reactions, thereby forming single-stranded linear DNA.
  • the special bases and specific sequences in the circular DNA molecule can be cut by adding appropriate enzymes, so that the single-stranded circular DNA can be opened.
  • the way of opening the ring is related to the special base and specific sequence in the circular DNA molecule.
  • the appropriate enzyme is selected according to the latter.
  • the schematic diagram of the ring opening is shown in Figure 2.
  • the fourth step perform reverse RCA reaction.
  • a reverse RCA reaction is performed using the single-stranded linear DNA as a primer to obtain an amplified double-stranded RCA product.
  • the single-stranded circular DNA is opened, the original single-stranded circular DNA molecule forms a single-stranded linear DNA molecule with an exposed 3' end, which can be recognized by polymerase.
  • the RCA reaction buffer and the required polymerase can be added, and the single-stranded linear DNA strand can be used as a primer to start the RCA reaction in the direction opposite to the initial RCA, that is, the reverse RCA reaction, as shown in Figure 2. It is easy to understand that the reverse RCA reaction does not require any additional primers or annealing, and the original template can be used as a primer for double-strand synthesis to obtain a complete RCA double-strand synthesis product in the same reaction system.
  • Example 1 Using DNA fragments as templates to verify the feasibility of the present invention.
  • PCR-1 To prepare template DNA-1, configure the following PCR-1 system: add 5 ⁇ L 10 ⁇ Standard Taq Reaction Buffer (NEB), 1 ⁇ L 10mM dNTPs (NEB), 0.25 ⁇ L Taq DNA Polymerase (NEB), 0.25 ⁇ M GAPDH500Fp-1 primer (Huada Liuhe), 0.25 ⁇ M GAPDH500R primer (Huada Liuhe), 0.01ng human transcriptome cDNA.
  • NEB 10 ⁇ Standard Taq Reaction Buffer
  • NEB 1 ⁇ L 10mM dNTPs
  • NEB 0.25 ⁇ L Taq DNA Polymerase
  • GAPDH500Fp-1 primer Humanada Liuhe
  • 0.25 ⁇ M GAPDH500R primer Human transcriptome cDNA
  • PCR product was purified with 0.8 ⁇ AMPure magnetic beads (Beckman) to obtain template DNA-1.
  • AMPure magnetic beads For the purification experimental steps, refer to the instructions of AMPure magnetic beads.
  • GAPDH500Fp-1 primer sequence (SEQ ID NO.1): 5'-Phosphate-AGCCACA U CGC U CAGACAC-3';
  • GAPDH500R primer sequence (SEQ ID NO.2): 5'-GAGGCATTGCTGATGATCTTG-3'.
  • PCR-2 To prepare template DNA-2, configure the following PCR-2 system: add 5 ⁇ L 10 ⁇ Standard Taq Reaction Buffer (NEB), 1 ⁇ L 10mM dNTPs (NEB), 0.25 ⁇ L Taq DNA Polymerase (NEB), 0.25 ⁇ M GAPDH500Fp to 50 ⁇ L reaction system -2 primer (Hua Da Liuhe), 0.25 ⁇ M GAPDH500R primer (Hua Da Liuhe), 0.01ng human transcriptome cDNA.
  • NEB 10 ⁇ Standard Taq Reaction Buffer
  • NEB 1 ⁇ L 10mM dNTPs
  • NEB 0.25 ⁇ L Taq DNA Polymerase
  • GAPDH500Fp to 50 ⁇ L reaction system -2 primer
  • Hua Da Liuhe Hua Da Liuhe
  • GAPDH500R primer Human transcriptome cDNA
  • PCR product was purified with 0.8 ⁇ AMPure magnetic beads (Beckman) to obtain template DNA-2.
  • AMPure magnetic beads For the purification experimental steps, refer to the instructions of AMPure magnetic beads.
  • GAPDH500Fp-2 primer sequence (SEQ ID NO.3): 5'-Phosphate-AGCCACA I CGC I CAGACAC-3';
  • GAPDH500R primer sequence (SEQ ID NO.4): 5'-GAGGCATTGCTGATGATCTTG-3'.
  • the GAPDH500Fp-1 primer used in this example was modified with a special base, and the special base dU was introduced at the linker through PCR reaction.
  • the GAPDH500Fp-2 primer used in this example was modified with a special base, and a special base I was introduced at the linker by PCR reaction.
  • step 4 Circularize the template DNA-1 and DNA-2 obtained in step 1, respectively.
  • configure the following reaction system add 12.5 ⁇ L 0.1M TE buffer, 2.5 ⁇ L GAPDH500splint (20 ⁇ M) primer and 330 ng of the above template DNA-1 or DNA-2 to the 48 ⁇ L reaction system. Mix well and place on a PCR instrument, incubate at 95°C for 3 minutes, and then immediately place on ice for further incubation for 10 minutes.
  • the GAPDH500splint primer sequence is (SEQ ID NO.7): 5'-AGCGATGTGGCTGAGGCATTGCTG-3'.
  • step 6 Purify the cyclization product in step 5 with 2.5 ⁇ AMPure magnetic beads (Beckman), and quantify it with Qubit ssDNA Kit.
  • Helicase is a DNA-binding protein that needs ATP (adenosine triphosphate) to provide energy, and different helicases have different directions. What this embodiment selects is the Tte UvrD helicase of NEB, which is a kind of ATP-dependent 3'-5' helicase. Tte UvrD helicase can spontaneously bind to single-strand DNA, and when there is no ATP present, the helicase has no unwinding effect. When ATP is added during the RCA process of this embodiment, the unwinding and RCA reactions proceed simultaneously, and the helicase will unwind along the 3' to 5' direction of the RCA product with the assistance of ATP. The introduction of helicase can make the RCA product produce as little secondary structure as possible.
  • ATP adenosine triphosphate
  • RCA reaction solution as follows: add 10 ⁇ L RCA buffer, 20 ⁇ L RCA enzyme mix1 and 2 ⁇ L RCA enzyme mix2 (MGIEasy stLFR library preparation kit) to the PCR tube, then add 4 ng (1) DNA-1 and DNA-2 product, and rehydrate to 37.5 ⁇ L respectively.
  • step 2 Set up a control experiment for the reaction in step 1 at the same time.
  • the reagents added in the control experiment were: 3 ⁇ L NEB buffer 2, and rehydrated to 30 ⁇ L. Place the reaction solution on a PCR instrument and incubate at 37°C for 30 minutes.
  • step 3 set up a control experiment for the reaction in step 3.
  • the reagents added in the control experiment are: 3 ⁇ L NEB buffer 4, and make up to 30 ⁇ L of water.
  • the resulting reaction solution was placed on a PCR instrument and incubated at 37°C for 30 minutes.
  • the DNA-1 reaction solution here uses the UDG/APE1 digestion scheme, because dU bases are introduced during the template preparation process; the DNA-2 reaction solution uses Endonuclease V for digestion, because the template During the preparation, the I base was introduced. For different bases or specific sequences, different ring-opening schemes need to be selected, which will not be repeated in this embodiment.
  • Reverse RCA reaction that is, RRCR ( Reverse Rolling Circle Replication ) reaction.
  • a reverse RCA reaction is performed using the single-stranded linear DNA as a primer to obtain an amplified double-stranded RCA product.
  • Example 2 Combining with the MGIEasy stLFR library preparation kit, the full-length mRNA transcripts were constructed and sequenced.
  • MGI's stLFR single tube Long Fragment Read
  • the average full length of human cDNA is about 2kb.
  • multiple copies of the full-length cDNA sequences are connected together, thereby realizing the preparation and enrichment of the full-length cDNA.
  • the capture sequence used to capture mRNA, the TSO primer used for reverse transcription, the ISO primer, the oligo dT sequence used for rolling circle amplification, and the TnSplint primer used for circularization and dissolve them respectively in TE solution to The concentration is 100 ⁇ M, and it is stored at -20 degrees Celsius for later use.
  • the input amount of 1 ⁇ g total RNA is taken as an example to carry out the following steps.
  • N refers to any one of the four bases A/T/C/G
  • V refers to any one of the three bases A/G/C
  • TSO primer (SEQ ID NO.9): 5'-AAGCdUdUCGTAGCCATGTCGTTCTGrGrGrG-3', wherein rG refers to the RNA base G, which is guanine ribonucleotide;
  • Oligo dT sequence (SEQ ID NO.11): 5'-TTTTTTTTTTTTTTTTTTTTTTTT-3'.
  • reverse transcriptase reaction mixture which contains 1 ⁇ L reverse transcriptase (SuperScript II reverse transcriptase (200U/ ⁇ L), Invitrogen Company), 0.5 ⁇ L RNaseOUT TM (RNase inhibitor, 40U/ ⁇ L, Invitrogen Company), 4 ⁇ L 5 ⁇ Superscript II first-strand buffer (5-fold concentration of reverse transcriptase II buffer; 250mM Tris-HCl, pH 8.3; 375mM KCl; 15mM MgCl 2 , Invitrogen Company), 0.5 ⁇ L DTT (100mM, Invitrogen Company), 6 ⁇ L MgCl 2 (25 mM, Invitrogen Company), 0.5 ⁇ L TSO primer (100 ⁇ M), make up the volume to 20 ⁇ L with water.
  • the obtained reverse transcription reaction system is placed in a PCR instrument for reverse transcription reaction, and the following procedures are run: (1) 90 minutes at 42 degrees Celsius; (2) 2 minutes at 50 degrees Celsius; (3) 2 minutes at 42 degrees Celsius; wherein (2) to (3) Run 10
  • transcript amplification reaction mixture including 50 ⁇ L 2 ⁇ KAPA HiFi HotStart Ready Mix (2 times concentration of KAPA HIFI hot-start enzyme mixture) (5mM MgCl 2 , Each dNTP 0.6mM, 1U KAPA HiFi HotStart DNA Polymerase, 5 ⁇ L ISO primer (10 ⁇ M), supplemented with water to a volume of 100 ⁇ L.
  • the amplification reaction system obtained was subjected to the amplification reaction according to the following procedures: (1) 98 degrees Celsius for 3 minutes; ( 2) 98 degrees Celsius for 20 seconds; (3) 67 degrees Celsius for 15 seconds; (4) 72 degrees Celsius for 6 minutes; (5) 72 degrees Celsius for 5 minutes; wherein steps (2) to (5) are repeated for 10-20 cycles.
  • the amplified product is purified with 200 ⁇ L of XP magnetic beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT Company).
  • XP magnetic beads Amincourt AMPure XP-Medium, A63882, AGENCOURT Company.
  • purification method refer to the standard operating procedures provided by the manufacturer.
  • step 4 After the purification in step 4 is completed, add 1 ⁇ L USER enzyme (1U/ ⁇ L NEB), 3 ⁇ L 10 ⁇ stTaq Buffer (10 times concentration of standard Taq buffer, 100 mM Tris-HCl, 500 mM KCl, 15 mM MgCl) to the purified product 2 ), make up the volume to 30 ⁇ L with water.
  • the resulting reaction system was placed in a PCR instrument at 37°C for 1 hour. At this point, the USER enzyme can cut out the cohesive ends on the cDNA to facilitate subsequent ligation and circularization.
  • step 5 After the reaction in step 5 is completed, add 5 ⁇ L 10 ⁇ TA Buffer to it, supplement the volume with water to 50 ⁇ L, place it in a PCR instrument at 70 degrees Celsius for 30 minutes, and then bathe in water at room temperature for 20 minutes.
  • step 6 After the reaction in step 6, add 2 ⁇ L 10 ⁇ TA Buffer, 0.752 ⁇ L 0.1M ATP, 0.1 ⁇ L T4 DNA Ligase (Enzymatics, 600U/ ⁇ L) to it, make up the volume to 55 ⁇ L with water, and react at room temperature for 2 hours.
  • reaction product is purified with 55 ⁇ L of XP magnetic beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT Company).
  • XP magnetic beads Amincourt AMPure XP-Medium, A63882, AGENCOURT Company.
  • step 8 After the purification in step 8, add 3 ⁇ L 10 ⁇ TA Buffer, 1.95 ⁇ L Exonuclease I (20 U/ ⁇ L, NEB M0293S), 0.65 ⁇ L Exonuclease III (100 U/ ⁇ L, NEB M0206S) to the purified product, and add water to the volume 30 ⁇ L, placed in a PCR instrument at 37 degrees Celsius for 30 minutes.
  • reaction product is purified with 60 ⁇ L of XP magnetic beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT Company).
  • XP magnetic beads Amincourt AMPure XP-Medium, A63882, AGENCOURT Company.
  • step 12 Add 20 ⁇ L of the rolling circle amplification reaction solution prepared in step 11 to the purified product in step 10, and make up the volume to 40 ⁇ L with water. Run the following program on the above reactants: 1 minute at 95 degrees Celsius, 1 minute at 65 degrees Celsius, and 1 minute at 40 degrees Celsius. After the program is over, immediately take out the product and place it on ice. At this time, the oligo dT primer anneals and binds to the purified product as a template.
  • step 13 After the reaction in step 13 is over, use a single-strand concentration detection kit (Lifetech) to detect the concentration.
  • step 15 Take 100ng of the product obtained in step 13, add 2 ⁇ L 10 ⁇ NEB buffer 4 (10 times the concentration of NEB buffer 4), 2 ⁇ L NEB Endonuclease V, and make up the volume to 20 ⁇ L with water, put it in a PCR machine and run the following program: 37 30 minutes at 65 degrees Celsius, 10 minutes at 65 degrees Celsius, 20 ⁇ L Make DNB Buffer (BGI) and 2 ⁇ L RCA Enzyme Mix (BGI) were added after the reaction, placed in a PCR machine, 30 degrees Celsius for 30 minutes, 65 degrees Celsius for 10 minutes.
  • BGI Make DNB Buffer
  • BGI 2 ⁇ L RCA Enzyme Mix
  • step 16 After the reaction in step 15, the obtained product was purified with 50 ⁇ L of XP magnetic beads (Agencourt AMPure XP-Medium, A63882, AGENCOURT Company). For the purification method, see the standard operating procedures provided by the manufacturer. So far, the preparation and enrichment of mRNA full-length transcripts (double-stranded cDNA) have been completed.
  • XP magnetic beads Amincourt AMPure XP-Medium, A63882, AGENCOURT Company
  • BGISEQ-500 for sequencing, the library established in 1 is required for single-strand circularization.
  • the circularization steps of the BGISEQ-500 standard DNA small fragment library construction process Through molecular label information, the short fragment information obtained by sequencing is restored to cDNA long fragment information, and the mRNA expression level is obtained.
  • Figure 6 shows the relationship between the assembled coverage length and the coverage ratio of Sample 1, wherein the relationship between the assembled coverage length and the coverage ratio is shown using a dot plot.
  • the x-axis represents the coverage length of the assembled contigs (contigs) on the transcript
  • the y-axis represents the coverage of the transcript by the contigs
  • the color from black to gray represents the length of the transcript from short to long.
  • Figure 7 shows the relationship between the transcript length and the assembly coverage of sample 1, wherein the relationship between the transcript length and the coverage is displayed using a dot plot.
  • the x-axis represents the length of the transcript
  • the y-axis represents the coverage of the transcript by the contig
  • the color from black to gray represents the assembly length from short to long. The results in Figure 7 show that most of the transcripts can be assembled to full length.
  • Figure 8 shows the distribution of coverage lengths assembled for Sample 1, using histograms to show the distribution of assembled contigs versus transcript coverage lengths. Among them, the x-axis represents the coverage length of the assembled contig to the transcript, and the y-axis represents the frequency. The results in Figure 8 show that the frequency of full-length contig assembly is high.

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Abstract

一种通过单链滚环扩增获得双链序列的方法。所述方法包括：1)以第一引物对单链环状DNA进行滚环扩增反应，获得扩增序列，所述第一引物与所述单链环状DNA部分区域互补，所述单链环状DNA具有可致所述单链环状DNA开环的断开机制；2)通过所述断开机制使所述单链环状DNA开环，获得单链线性DNA；3)以所述单链线性DNA为第二引物、以1)中获得的扩增序列为模板进行扩增反应，获得扩增的双链序列。该方法通过单链滚环扩增获得双链序列具有诸多优点。

Description

一种通过单链滚环扩增获得双链序列的方法 技术领域
本发明涉及生物技术领域，更具体而言，涉及一种通过单链滚环扩增获得双链序列的方法。
背景技术
滚环扩增(Rolling Circle Amplification，RCA)或滚环复制(Rolling Circle Replication，RCR)反应通常是指使用具有链置换活性的DNA聚合酶(例如Phi29聚合酶、Bst聚合酶、Bsu聚合酶、Klenow Fragment、Vent聚合酶、Pol III聚合酶等)，以单链环状DNA分子为模板进行的聚合酶链式反应。通常，在合适的条件下，当加入一段与某单链环状DNA分子序列上互补配对的DNA或RNA片段时，具有链置换活性的DNA聚合酶就会以单链环状DNA为模板，以加入的DNA或RNA片段作为引物，对模板DNA进行扩增反应。因为这种DNA聚合酶不具有5’→3’的外切酶活性，所以当扩增完成一个循环后，聚合酶会将前进方向的DNA双链的一条解旋，继续反应，从而得到含有多个相同拷贝的单链DNA分子。同理的，当环状双链DNA分子(例如质粒、病毒DNA分子等)的一条链上有断裂或碱基缺失时，上述滚环扩增同样可以发生。
由具有链置换活性的聚合酶参与的滚环扩增反应，因其反应速度快、保真度高且反应温度恒定等优点，也常常被称为滚环等温扩增反应，并得到广泛应用。同理的，非环状DNA分子同样可以基于上述原理进行等温扩增反应。当滚环扩增反应时间足够持久，生成的DNA单链会相互折叠缠绕，形成复杂的空间二级结构，与金属离子相互配位形成结构致密的DNA分子纳米球(DNA Nanoball，简称DNB)，也称为DNP(DNA Nanoparticle)、DNF(DNA Nanoflower)等。DNB具有许多常规DNA分子所不具有的性质，例如抗DNase消化、与金属离子形成胶体、有较高的细胞亲和力等。
DNB虽然具有上述优势，但是其主要构成是单链DNA分子，RCA产物在稳定性和酶反应的多样性方面，相较于双链DNA分子，仍然具有很大的局限性。因此，将单链的RCA产物转化为双链的产物，将极大地拓展RCA的应用，也可以通过与其他技术联合应用形成互补效应，促进技术升级。
RCA产物双链化的几种现有方案简述如下：
1)RCA反应的副反应：目前在RCA反应中被广泛应用的扩增酶为Phi29及其突变体。Phi29具有很强的链置换活性，且具有一定的模板跳转(Template Switch)反应活性，即在反应过程中，当Phi29处于复制叉的位置时，特别是在反应初始阶段，其有一定的概率会置换到附近的单链DNA上，并以此DNA为模板，向与RCA初始方向相反的方向进行复制，最终形成一段双链DNA产物。然而，该RCA产物虽然为双链产物，但是属于RCA反应的副产物，一般产量较低。同时，因为该副反应往往发生在反应的初始阶段。因此，所得到的双链RCA产物长度较短，扩增倍数也较少，该产物无法被有效利用。
2)双引物RCA反应：双引物RCA，即在RCA反应中加入与DNA单链环互补配对的反向引物和与模板序列一致的正向引物，同时进行RCA反应。反应体系中与模板序列互补配对的反向引物会首先结合在DNA单链环上进行RCA反应，随后与模板序列一致的正向引物会结合在生成的RCA产物上，以RCA产物为模板，在Phi29的作用下继续延伸，进而形成双链RCA产物，这个过程被称之为DNB的二链合成。同理的，随着Phi29的继续延伸，新生成的二链产物的5’末端会被置换下来，体系中的反向引物会结合在置换出的二链产物上，在Phi29的作用下继续延伸，如此往复，形成复杂的双链DNA网状结构。双引物RCA反应，是最常用的一种等温扩增体系。该体系的优势是可以在短时间内，生成大量双链DNA拷贝，获得相对稳定的扩增产物。然而，这种方法的缺点也很明显，由于加入各个引物的量难以精确控制，RCA反应会持续地在不同的支链中反复进行，一方面会促使整个双链DNB的结构异常复杂；另一方面，因为空间结构的限制，很多二链合成无法进行完全，生成一个单双链同时存在的DNB结构，给定性和定量都增加了难度。
3)随机引物RCA反应(R2C2，Rolling Circle Amplification to Concatemeric Consensus Method)：随机引物法与双引物法类似，区别在于随机引物可以结合在单链RCA产物的任何位置，随机引物法获得的双链RCA产物会更加复杂，二链合成相比双引物RCA法更完全。反应中需要用到外切酶，对生成的多余单链DNA进行消化，保证尽可能多的双链生成。该方法虽然可以获得较多完整的双链RCA产物，但是因为随机引物结合的位点太多，会形成很多支链结构，与常规的链状DNA分子还是有一定的区别，性质上也不完全一致。
4)环介导等温扩增(Loop-mediated isothermal amplification，LAMP)法RCR反应：在单链DNA分子两端加上具有互补配对序列的接头，并形成一个茎环结构。进行一步等温扩增后将单链转化为双链结构，再加入另一个与茎环结构互补配对的引物进行延伸，将双链结构打开。被打开的单链3’端接头会发生分子内折叠，再次形成茎环结构，通过一步等温扩增，即可得到一个具有两份拷贝的双链DNA分子。不断重复该过程即可得到一个具有重复序列的长片段双链DNA分子。目前LAMP法是通过等温扩增获得大量多拷贝长片段分子的主要方法。该方法具有速度快，双链转化完全，副产物少等优势。但是因为反应过程中需要加入较多的接头、标签序列等，所以最后生成的分子中会存在较多冗余信息。
5)发卡接头扩增法(Hairpin RPA Amplification)：是上海灏勤生物科技有限公司提出的一种可以将短分子进行自组装形成长多聚体分子的方法，与LAMP类似但实现方式更加简便。该方法是将带有(…TATATATA…)回文序列的接头连接在目的片段的两端，因为TA序列的退火温度较低，在温度逐渐上升的接头部分会发生解链，回文序列在温度合适的情况下，会发生分子内配对的特性(即DNA呼吸)，发生折叠和配对的3’末端会作为新的延伸位点，在扩增酶的作用下进行分子内延伸，从而将原分子长度扩展一倍。不断重复这一步骤，即可获得含有多拷贝的长片段双链DNA分子。该方法相比LAMP，发卡接头扩增法的接头设计更加简单，缺点在于DNA呼吸的温度控制需要调试，TA回文序列会产生非特异性扩增产物。
总之，上述现有技术存在以下缺点：1)二链合成不完全：现有的多种RCA方案，都存在二链合成不完全的情况，所得到的双链RCA产物大都存在一些单链的Gap区域；2)二级结构复杂：上述几种主要的二链合成方法中，因为加入引物的量无法准确控制，且都需要较长的反应时间，因此生成的双链RCA产物往往具有复杂的二级结构，虽然本质上已经形成双链DNA结构，但是因为结构复杂，存在较多分支以及单链Gap区域，所以在生化反应的表现上与常规的双链DNA分子有所差异；3)副产物较多：如前所述，因为反应中需要加入外源的引物、外切酶、多种接头序列等因素，所以反应体系中会积蓄较多副产物，可能会干扰下游的实验设计。
发明内容
为了至少部分地解决现有技术RCA二链合成中存在的二链转化不完全、二级结构复杂、副产物多的问题，本发明提供了一种简易的获得双链RCA产物的解决方案。
因此，在第一方面，本发明提供了一种通过单链滚环扩增获得双链序列的方法，所述方法包括：
1)以第一引物对单链环状DNA进行滚环扩增反应，获得扩增序列，所述第一引物与所述单链环状DNA部分区域互补，所述单链环状DNA具有可致所述单链环状DNA开环的断开机制；
2)通过所述断开机制使所述单链环状DNA开环，获得单链线性DNA；
3)以所述单链线性DNA为第二引物、以1)中获得的扩增序列为模板进行扩增反应，获得扩增的双链序列。
在一个实施方案中，所述单链环状DNA来自对DNA样本或cDNA样本进行环化，通过PCR或接头连接，在所述单链环状DNA中引入特殊碱基或特异性序列。
在一个实施方案中，所述第一引物为DNA引物或RNA引物。
在一个实施方案中，可致所述单链环状DNA开环的断开机制为所述单链环状DNA包含特定区域，所述特定区域在生化反应下发生断裂，从而导致所述单链环状DNA开环。
在一个实施方案中，所述特定区域包括特殊碱基和/或特异性序列。
在一个实施方案中，所述特殊碱基为次黄嘌呤脱氧核苷酸(I)、脱氧尿嘧啶核苷酸(dU)、RNA碱基、AP位点或甲基化位点。
在一个实施方案中，所述特异性序列为限制性内切酶识别位点或蛋白质的特异性结合位点。
在一个实施方案中，所述限制性内切酶识别位点为富含AT碱基序列的区域；优选地，所述限制性内切酶识别位点为Chlamydomonas内切酶识别位点或Neurospora crassa内切酶识别位点。
在一个实施方案中，所述蛋白质的特异性结合位点为CRISPR/Cas基因编辑系统中向导RNA的识别区域；优选地，为CRISPR/Cas9中向导RNA的识别区域。
在一个实施方案中，在对单链环状DNA进行滚环扩增反应过程中或之后加入单链DNA结合蛋白、焦磷酸酶和TE缓冲液。
在一个实施方案中，在对单链环状DNA进行滚环扩增反应过程中或之后加入解旋酶。
在一个实施方案中，所述解旋酶为具有3’到5’方向解旋能力的A型解旋酶，优选为Rep解旋酶、UvrD解旋酶、Heli308解旋酶、PcrA解旋酶、或RecD2解旋酶。
在第二方面，本发明提供了一种核酸测序文库的构建方法，所述方法包括：
1)通过本发明第一方面所述的方法获得扩增的双链序列；
2)将所述双链序列进行测序文库构建，获得所述核酸测序文库。
在一个实施方案中，使用LFR技术(长片段读取技术)进行核酸测序文库构建，获得所述核酸测序文库。
在一个实施方案中，所述核酸测序文库是mRNA全长转录本文库。
在第三方面，本发明提供了一种测序方法，所述方法包括：
1)通过本发明第一方面所述的方法获得扩增的双链序列，或者本发明第二方面所述的方法获得核酸测序文库；
2)对所述扩增的双链序列或核酸测序文库进行测序。
在一个实施方案中，所述测序为高通量测序。
在一个实施方案中，所述高通量测序为第二代测序或第三代测序。
本发明的方法有如下优点：扩增反应无需加入外源引物，副产物较少；生成的双链RCA产物单一、无支链；生成的双链RCA产物易捕获、易纯化；反应为单管反应、操作简单易行；解旋酶辅助降低二级结构的复杂性、产物更接近常规DNA分子结构；生成的完整长片段双链多拷贝DNA可以应用于单分子测序，特别是ONT平台，多拷贝DNA分子有利于提高单分子测序的准确率。
附图说明
图1分别示出了带有特殊碱基或特异性序列的单链环状DNA示意图(A)和常规RCA反应产物示意图(B)。
图2示意性示出了本发明的通过单链滚环扩增获得双链序列的方法。
图3示出了3’-5’方向的解旋酶与滚环扩增序列结合的原理示意图。
图4示出了解旋酶对二链合成效率的影响。
图5示出了不同二级结构复杂的RCA产物的琼脂糖凝胶电泳结果。
图6示出了样品1组装的覆盖长度与覆盖率的关系。
图7示出了样品1转录本长度与组装覆盖率的关系。
图8示出了样品1组装的覆盖长度分布。
具体实施方式
本发明提出了一种简易的获得双链RCA产物的方法，首先以第一引物对单链环状DNA进行滚环扩增反应获得扩增序列，然后通过断开机制(例如所述单链环状DNA上的特殊碱基或特异性序列)将所述单链环状DNA环模板进行开环，获得单链线性DNA；最后，以所述单链线性DNA为引物进行反向RCA反应，获得扩增的双链RCA产物。
在一个具体实例中，本发明方法可以包括四个步骤：第一步骤包括制备带有能够使所述单链环状DNA开环的特殊碱基(或特异性序列)的单链环状DNA；第二步骤包括以第一引物对单链环状DNA进行RCA反应获得扩增序列，在RCA反应过程中或者在RCA反应之后可以加入解旋酶，使之参与所述RCA反应；第三步骤包括通过生化反应，将所述单链环状DNA在特殊碱基或特异性序列位置上产生断裂，进而形成单链线性DNA；第四步骤包括以所述单链线性DNA为引物进行反向RCA反应，获得扩增的双链RCA产物。图2示意性示出了本发明的通过单链滚环扩增获得双链序列的方法，以下分步骤示例性描述本发明的方法的原理：
第一步骤：制备带有特殊碱基(或特异性序列)的单链环状DNA。
在本发明中，可以对DNA样本直接环化，也可以用总RNA逆转录得到cDNA后再进行环化制备单链环状DNA。可以通过PCR、接头连接等方法，在单链环状DNA中引入特殊碱基或特异性序列，从而在需要时通过生化反应，所述单链环状DNA在特殊碱基或特异性序列位置处发生断裂，进而导致所述单链环状DNA开环，获得单链线性DNA链。在图1中，A示出了带有特殊碱基或特异性序列的单链环状DNA示意图。所述特殊碱基包括但不限于I碱基(次黄嘌呤核苷酸)、dU碱基(尿嘧啶脱氧核苷酸)、RNA碱基、AP位点、甲基化位点等；也可以引入特异性序列，包括但不限于限制性内切酶识别位点等。在 实施例以I碱基和dU碱基为例，实现开环反应，其他开环方式在此不一一列举。
在本发明中，所述特殊碱基或特异性序列将所述单链环状DNA开环优选是受控的，例如在适合生化反应的条件下实现所述单链环状DNA开环。例如：
1)对于次黄嘌呤核苷酸，即I碱基，可使用核酸内切酶V进行消化切除，从而使单链环打开；
2)dU碱基可以使用UDG酶或APE1酶进行识别和切除，从而使单链环打开；
3)RNA碱基可以使用RNaseA，RNaseH等酶进行识别和切除，从而使单链环打开；
4)AP位点可以使用APE1酶进行识别和切除，从而使单链环打开；
5)甲基化的C碱基位点，可以使用亚硫酸氢钠处理或者用APOBEC脱氨酶或TET2酶进行处理，将甲基化C碱基转化为dU碱基，再通过UDG酶或APE1酶进行识别和切除，从而使单链环打开；
6)对于单链环上的富TA序列，可以使用Chlamydomonas内切酶或Neurospora crassa内切酶进行识别和切除，从而使单链环打开；
7)在单链环中引入特异性的蛋白结合序列(在一个实例中具体序列为CRISPR/Cas基因编辑系统中导向RNA的识别序列)，通过利用含突变的CRISPR/Cas9系统，在DNA分子上产生单链缺口SSB(Single-Stranded Break)而使单链环开环。
除此上述以外任何可以使单链DNA开环的碱基、特异性序列均可作为该方案的备选方案，为后续DNA的开环反应做准备。本发明意欲涵盖任何可以使单链DNA开环的碱基与特异性序列。
第二步骤：进行RCA反应。
以第一引物对单链环状DNA进行RCA反应获得扩增序列。RCA反应速率非常快，形成的单链DNA在焦磷酸及镁离子的作用下，容易相互退火、缠绕、折叠形成复杂的二级结构。在图1中，B示出了常规RCA反应产物示意图。为了使生成的RCA产物结构相对松散，优选在RCA反应中添加单链DNA结合蛋白、焦磷酸酶和TE缓冲液等组分，用来获得相对“蓬松”的RCA产物。然而，即使加入上述试剂，所得到的RCA产物最终仍然会形成致密的DNB形态分子，这样不利于二链合成。一方面RCA反应后投入的二链引物很难完全结合到RCA 产物上，另一方面，因为空间位阻等因素，二链合成酶无法将RCA产物转化为完整的双链产物。
在本发明中，在一个优选实施方案中，优选可以加入解旋酶，使之参与RCA反应，可以达到更好的RCA产物“蓬松”效果。图3示出了3’-5’方向的解旋酶与滚环扩增序列结合的原理示意图。在本发明中，可以在RCA反应过程中或者在RCA反应之后加入解旋酶。不希望拘囿任何具体的理论，解旋酶利用ATP水解释放的能量，发生构象变化，同时能够打开双链DNA的氢键结构。解旋酶会特异性的结合在单链DNA上，并在ATP的作用下开始从3’到5’方向进行解旋反应。在RCA反应过程中或者在RCA反应之后加入解旋酶可以帮助RCA产物保持相对简单的单链形式，为后续的反向RCA反应创造条件。
解旋酶具有方向性，本发明采用3’到5’方向的单链DNA解旋酶(即A型α解旋酶系列；Type Aα-Helicase Family)作为实施例来阐释本发明的原理，如图3所示。在具体实例中，本发明可以采用NEB的Tte UvrD解旋酶。同理的，任何具有3’到5’方向解旋能力的A型解旋酶，例如Rep、Heli308、PcrA、RecD2等，均可作为本实施例的备选方案，本发明意欲包括这些解旋酶。
第三步骤：使得单链环状DNA开环。
在本发明中，通过生化反应，将所述单链环状DNA在特殊碱基、特异性序列位置上产生断裂，进而形成单链线性DNA。例如，在经过一段时间的RCA反应后，可以通过加入适当的酶，将环状DNA分子中的特殊碱基、特异性序列进行剪切，从而使单链环状DNA开环。开环方式与环状DNA分子中的特殊碱基、特异性序列有关，例如根据后者选用适当的酶，开环示意图如图2所示。容易理解的，当单链环状DNA发生开环后，参与RCA反应的聚合酶(如Phi29)到达缺口位置时，因为失去了模板而从模板分子上掉落，从而终止了RCA反应的继续进行。
第四步骤：进行反向RCA反应。
在该步骤中，以所述单链线性DNA为引物进行反向RCA反应，获得扩增的双链RCA产物。当单链环状DNA开环后，原来的单链环状DNA分子形成的单链线性DNA分子具有裸露的3’末端，该末端可以被聚合酶识别。此时补加RCA的反应缓冲液及所需的聚合酶，可以以单链线性DNA链为引物，开始向与初始RCA相反的方向进行RCA反应，即反向RCA反应，如图2所示。容易 理解的，该反向RCA反应不需要额外加入任何引物，也无需退火，利用原始模板即可作为二链合成的引物，在同一反应体系中获得完整的RCA双链合成产物。
实施例一：以DNA片段为模板，验证本发明的可行性。
(一)、制备模板DNA。
1.为了制备模板DNA-1，配置如下PCR-1体系：于50μL反应体系中加入5μL 10×Standard Taq Reaction Buffer(NEB)、1μL 10mM dNTPs(NEB)、0.25μL Taq DNA Polymerase(NEB)、0.25μM GAPDH500Fp-1引物(华大六合)、0.25μM GAPDH500R引物(华大六合)、0.01ng人转录组cDNA。将得到的PCR混合液置于PCR仪上，运行如下程序：98摄氏度2分钟；重复(95摄氏度30秒、56摄氏度30秒、72摄氏度2分钟)20个循环；最后72摄氏度孵育10分钟，并降至4摄氏度保存。反应结束后用0.8×AMPure磁珠(Beckman)对PCR产物进行纯化获得模板DNA-1，纯化实验步骤参考AMPure磁珠说明书。
GAPDH500Fp-1引物序列(SEQ ID NO.1)：5’-Phosphate-AGCCACA UCGC UCAGACAC-3’；
GAPDH500R引物序列(SEQ ID NO.2)：5’-GAGGCATTGCTGATGATCTTG-3’。
为了制备模板DNA-2，配置如下PCR-2体系：于50μL反应体系中加入5μL 10×Standard Taq Reaction Buffer(NEB)、1μL 10mM dNTPs(NEB)、0.25μL Taq DNA Polymerase(NEB)、0.25μM GAPDH500Fp-2引物(华大六合)、0.25μM GAPDH500R引物(华大六合)、0.01ng人转录组cDNA。将得到的PCR混合液置于PCR仪上，运行如下程序：98摄氏度2分钟；重复95摄氏度30秒、56摄氏度30秒、72摄氏度2分钟20个循环；最后72摄氏度孵育10分钟，并降至4摄氏度保存。反应结束后用0.8×AMPure磁珠(Beckman)对PCR产物进行纯化获得模板DNA-2，纯化实验步骤参考AMPure磁珠说明书。
GAPDH500Fp-2引物序列(SEQ ID NO.3)：5’-Phosphate-AGCCACA ICGC ICAGACAC-3’；
GAPDH500R引物序列(SEQ ID NO.4)：5’-GAGGCATTGCTGATGATCTTG-3’。
2.本例中使用的GAPDH500Fp-1引物被特殊碱基进行修饰，通过PCR反应，在接头处引入了特殊碱基dU。本例中使用的GAPDH500Fp-2引物被特殊碱基进 行修饰，通过PCR反应，在接头处引入了特殊碱基I。使用这类方法，同样也可以在模板DNA上引入包括但不限于AP位点、甲基化位点、特异性序列等。
3.经纯化，得到的模板DNA-1序列如下(SEQ ID NO.5)：
Figure PCTCN2021100436-appb-000001
经纯化，得到的模板DNA-2序列如下(SEQ ID NO.6)：
Figure PCTCN2021100436-appb-000002
4.将步骤1中获得的模板DNA-1和DNA-2分别进行环化。为了进行环化，配置如下反应体系：在48μL反应体系中加入12.5μL 0.1M TE缓冲液、2.5μL GAPDH500splint(20μM)引物和330ng上述模板DNA-1或DNA-2。充分混匀后置于PCR仪上，95摄氏度孵育3分钟，然后立刻置于冰上继续孵育10分钟。向上述48μL反应体系中加入6μL 10×T4 DNA ligation buffer(NEB M0202S)、0.6μL 100mM ATP(NEB P0756S)、0.2μL T4 DNA ligase(600U/μL)(NEB M0202S)，并补水至总体积为60μL。将反应液置于PCR仪上，37摄氏度孵育1个小时。
GAPDH500splint引物序列为(SEQ ID NO.7)： 5’-AGCGATGTGGCTGAGGCATTGCTG-3’。
5.取步骤4中DNA-1和DNA-2的反应液各4μL至新的PCR管中备用，向剩余56μL反应液中加入0.4μL 10×T4 DNA ligation buffer(NEB M0202S)、1.95μL Exonuclease I(20U/μL，NEB M0293S)、0.65μL Exonuclease III(100U/μL，NEB M0206S)，并补水至60μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。
6.用2.5×AMPure磁珠(Beckman)纯化步骤5中的环化产物，并用Qubit ssDNA Kit进行定量。
(二)、解旋酶参与的RCA反应(Heli-RCA)。
1.解旋酶是一种需要ATP(三磷酸腺苷)供能的DNA结合蛋白，且不同的解旋酶具有不同的方向性，本实施例选用的是NEB的Tte UvrD解旋酶，这是一种ATP依赖的3’-5’解旋酶。Tte UvrD解旋酶可以自发地结合在DNA单链上，当没有ATP存在时，解旋酶没有解旋作用。在本实施例的RCA的过程中加入ATP，则解旋与RCA反应同时进行，解旋酶会在ATP的协助下，沿着RCA产物的3’到5’方向进行解旋。引入解旋酶可以使RCA产物尽可能少的产生二级结构。
2.配置如下RCA反应液：向PCR管中加入10μL RCA buffer、20μL RCA enzyme mix1和2μL RCA enzyme mix2(MGIEasy stLFR文库制备试剂盒)，然后分别加入4ng(一)步骤6中的DNA-1和DNA-2产物，并分别补水至37.5μL。将得到的反应液置于PCR仪上，30摄氏度孵育5分钟后立刻置于冰上，然后分别加入0.5μL Tte UvrD解旋酶(13.4μM)和2μL ATP(0.1M)(NEB)，在充分混匀后，置于PCR仪上，30摄氏度孵育25分钟，升温至65摄氏度，继续孵育15分钟。
3.同时设置一组对照试验，反应条件及配方相似，不同之处是将上述实验中的Tte UvrD解旋酶替换为等量的分子级水即可。
(三)、开环反应。
1.取20μL(二)中RCA反应后得到的DNA-1反应液至新的PCR管中，向其中加入如下试剂：3μL NEB buffer 2、2μL UDG(5U/μL，NEB)、3μL APE1(10U/μL，NEB)，并补水至30μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。同时取20μL(二)中RCA反应后得到的DNA-1对照组反应 液至新的PCR管中，向其中加入如下试剂：3μL NEB buffer 2、2μL UDG(5U/μL，NEB)、3μL APE1(10U/μL，NEB)，并补水至30μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。
2.同时针对步骤1中的反应设置对照试验。在对照试验中加入的试剂是：3μL NEB buffer 2，并补水至30μL。将反应液置于PCR仪上，37摄氏度孵育30分钟。
3.取20μL(二)中RCA反应后得到的DNA-2反应液至新的PCR管中，向其中加入如下试剂：3μL NEB buffer 4、1μL Endonuclease V(10U/μL，NEB)，并补水至30μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。同时取20μL(二)中RCA反应后得到的DNA-2对照组反应液至新的PCR管中，向其中加入如下试剂：3μL NEB buffer 4、1μL Endonuclease V(10U/μL，NEB)，并补水至30μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。
4.同时针对步骤3中的反应设置对照试验，在对照试验中加入的试剂是：3μL NEB buffer 4，并补水至30μL。将得到的反应液置于PCR仪上，37摄氏度孵育30分钟。
5.这里的DNA-1反应液使用了UDG/APE1的消化方案，这是因为在模板制备过程中，引入了dU碱基；DNA-2反应液使用了Endonuclease V进行消化，这是因为在模板制备过程中，引入了I碱基。针对不同的碱基或者特异性序列，需要选择不同的开环方案，本实施例中将不再赘述。
(四)、反向RCA反应，即RRCR( Reverse  Rolling  Circle  Replication)反应。
以所述单链线性DNA为引物进行反向RCA反应，获得扩增的双链RCA产物。取(三)中开环后得到的DNA-1和DNA-2的产物反应液，以及对应的对照试验的产物反应液，向其中分别加入如下试剂：20μL RCA enzyme mix1和2μL RCA enzyme mix2(MGIEasy stLFR文库制备试剂盒)充分混匀后，置于PCR仪上，30摄氏度孵育30分钟，升温至65摄氏度，孵育15分钟后4摄氏度保存。
实验结果：
上述实验过程可以简要表示为如下的步骤1至步骤5，共获得8种反应产物，分别标记为产物1到产物8。
Figure PCTCN2021100436-appb-000003
结果如下图4所示，产物1-8的dsDNA浓度(ng/μL)分别为21.30、12.76、9.33、9.87、14.38、11.55、6.42和8.96。由于有解旋酶参与的RCA反应，其二级结构相较于常规RCA反应更加简单，因此进行反向RCA时，二链合成更加彻底，对应的双链RCA产物的产量也更高。因此，上述结果显示，解旋酶的加入可以显著提高二链合成的效率。
对于正常的RCA产物和RCA二链合成产物，它们的二级结构复杂，在琼脂糖凝胶电泳时，产物会滞留在胶孔中，同时会有少量的DNA片段跑出胶孔形成弥散条带。对于线性双链RCA产物，其结构类似于通常的链状DNA分子，在琼脂糖凝胶电泳时，可以跑出胶孔。因此，可以通过琼脂糖凝胶电泳，直观的观察其二链合成情况，如图5所示。
实施例二：结合MGIEasy stLFR文库制备试剂盒，对mRNA全长转录本进行建库测序。
(一)、mRNA全长转录本(cDNA)的制备与富集。
由于MGI的stLFR(single tube Long Fragment Read，单管长片段序列)技术可实现读取序列的长度为10k～300k，而人源cDNA全长平均约为2kb。本实施例通过使用本发明提出的RCA扩增方法，将多拷贝的cDNA全长序列连在一起，进而实现全长cDNA的制备与富集。
1.合成用于捕获mRNA的捕获序列、用于逆转录的TSO引物、ISO引物、用于滚环扩增的oligo dT序列、用于环化的TnSplint引物，将它们分别用TE溶液回溶至浓度100μM，储存于-20摄氏度备用。本实施例以1μg总RNA投入量为例进行以下步骤。
捕获序列(SEQ ID NO.8)：
5'-AAGCdUdUCGTAGCCATGTCGTTCTGCGNNNNNNNNNNTTTTTTTTTTTTTTTTTTTTTV-3'，其中N指的是A/T/C/G四种碱基的任意一种，V指的是A/G/C 三种碱基的任意一种；
TSO引物(SEQ ID NO.9)：5’-AAGCdUdUCGTAGCCATGTCGTTCTGrGrGrG-3’，其中rG指的是RNA碱基G，即鸟嘌呤核糖核苷酸；
ISO引物(SEQ ID NO.10)：5‘-AAGCdUdUCGTAGICATGTIGTTCTG-3’；
oligo dT序列(SEQ ID NO.11)：5’-TTTTTTTTTTTTTTTT-3’。
2.取1μL人源总RNA(1微克)，加入5μL dNTP(10mM)、1μL捕获序列(50μM)，置于PCR仪中72摄氏度3分钟，立刻取出冰上放置1分钟。然后，加入逆转录酶反应混合液，其中包含1μL逆转录酶(SuperScript II reverse transcriptase(200U/μL)，Invitrogen公司)、0.5μL RNaseOUT TM(RNA酶抑制剂、40U/μL，Invitrogen公司)、4μL 5×Superscript II first-strand buffer(5倍浓度的逆转录酶II缓冲液；250mM Tris-HCl，pH 8.3；375mM KCl；15mM MgCl 2，Invitrogen公司)、0.5μL DTT(100mM，Invitrogen公司)、6μL MgCl 2(25mM，Invitrogen公司)、0.5μL TSO引物(100μM)，用水补足体积至20μL。将得到的逆转录反应体系置于PCR仪中进行逆转录反应，运行如下程序：(1)42摄氏度90分钟；(2)50摄氏度2分钟；(3)42摄氏度2分钟；其中(2)至(3)运行10个循环。
3.上述逆转录反应结束后，向其中加入全长转录本扩增反应混合液，其中包括50μL 2×KAPA HiFi HotStart Ready Mix(2倍浓度的KAPA HIFI热启动酶混合液)(5mM MgCl 2，每种dNTP 0.6mM，1U KAPA HiFi HotStart DNA Polymerase、5μL ISO引物(10μM)，用水补充体积至100μL。将得到的扩增反应体系按以下程序进行扩增反应：(1)98摄氏度3分钟；(2)98摄氏度20秒；(3)67摄氏度15秒；(4)72摄氏度6分钟；(5)72摄氏度5分钟；其中步骤(2)至(5)重复10-20个循环。
4.步骤3的扩增反应结束后，将扩增产物用200μL XP磁珠(Agencourt AMPure XP-Medium，A63882，AGENCOURT公司)进行纯化，纯化方法见厂商提供的标准操作规程。
5.步骤4的纯化结束后，向纯化后产物中加入1μL USER酶(1U/μL NEB)，3μL 10×stTaq Buffer(10倍浓度的标准Taq缓冲液、100mM Tris-HCl、500mM KCl、15mM MgCl 2)，用水补充体积至30μL。将得到的反应体系置于PCR仪 中37摄氏度反应1小时。此时，USER酶可在cDNA上切出粘性末端，以便于后续的连接环化。
6.步骤5的反应结束后，向其中加入5μL 10×TA Buffer，用水补充体积至50μL，将其置于PCR仪中70摄氏度反应30分钟，然后室温水浴20分钟。
7.步骤6的反应结束后，向其中加入2μL 10×TA Buffer、0.752μL 0.1M ATP、0.1μL T4 DNA Ligase(Enzymatics，600U/μL)，用水补充体积至55μL，在室温反应2小时。
8.步骤7的反应结束后，将反应产物用55μL XP磁珠(Agencourt AMPure XP-Medium，A63882，AGENCOURT公司)进行纯化，纯化方法见厂商提供的标准操作规程。
9.步骤8的纯化结束后，向纯化产物中加入3μL 10×TA Buffer、1.95μL Exonuclease I(20U/μL，NEB M0293S)、0.65μL Exonuclease III(100U/μL，NEB M0206S)，用水补充体积至30μL，置于PCR仪中37摄氏度反应30分钟。
10.在步骤9的反应结束后，将反应产物用60μL XP磁珠(Agencourt AMPure XP-Medium，A63882，AGENCOURT公司)进行纯化，纯化方法见厂商提供的标准操作规程。
至此完成了全长转录本的单链环化。
11.配制滚环扩增反应液，取4μL oligo dT(50μM)，加入40μL 10×phi29buffer(10倍浓度的phi29缓冲液)，用水补充体积至200μL。
12.向步骤10的纯化产物中加入20μL的步骤11中配制的滚环扩增反应液，用水补足体积至40μL。对上述反应物运行如下程序：95摄氏度1分钟，65摄氏度1分钟，40摄氏度1分钟，程序结束后，马上取出产物放置冰上，此时，oligo dT引物退火与作为模板的纯化产物结合。
13.向步骤12的产物中加入40μL Make DNB Buffer(MGI，P093)，4μL RCA Enzyme Mix(MGI，P094)，置于PCR仪中，30摄氏度2分钟，立即取出至于冰上，加入1μL Tte UvrD解旋酶(NEB，M1202S)、1μL ATP(10mM)，于PCR仪上进行反应：30摄氏度30分钟，65摄氏度10分钟。
14.步骤13的反应结束后，用单链浓度检测试剂盒(Lifetech公司)进行浓度检测。
15.取步骤13获得的产物100ng，加入2μL 10×NEB buffer 4(10倍浓度的NEB缓冲液4)、2μL NEB Endonuclease V，并用水补足体积至20μL，置于PCR仪中运行如下程序：37摄氏度30分钟，65摄氏度10分钟，反应结束后加入20μL Make DNB Buffer(BGI)、2μL RCA Enzyme Mix(BGI)，置于PCR仪中，30摄氏度30分钟，65摄氏度10分钟。
16.在步骤15的反应结束后，将得到的产物用50μL XP磁珠(Agencourt AMPure XP-Medium，A63882，AGENCOURT公司)纯化，纯化方法见厂商提供的标准操作规程。至此完成了mRNA全长转录本(双链cDNA)的制备与富集。
(二)、制备带有分子标签的短片段分子并进行测序。
1.使用MGIEasy stLFR文库制备试剂盒对第(一)中得到的mRNA全长转录本(双链cDNA)进行LFR建库，建库流程遵照MGIEasy stLFR试剂盒说明书进行。
2.使用BGISEQ-500进行测序，需要1中建立的文库进行单链成环反应，操作详情请参照BGISEQ-500标准DNA小片段建库流程的成环步骤。通过分子标签信息，将测序得到的短片段信息还原为cDNA长片段信息，获得mRNA表达量。
3.实验结果：
(1)测序结果如下表所示：
表一：测序读长结果统计
Figure PCTCN2021100436-appb-000004
(2)测序读长组装结果：
图6示出了样品1组装的覆盖长度与覆盖率的关系，其中使用点图展示组装的覆盖长度与覆盖率的关系。其中，x轴表示组装的重叠群(contigs)对转录本 的覆盖长度，y轴表示重叠群对转录本的覆盖率，颜色从黑色到灰色表示转录本长度从短到长。图6中的结果表明，各转录本的组装长度可以达到100％，最长的转录本覆盖长度可以达到约4000bp。
图7示出了样品1转录本长度与组装覆盖率的关系，其中使用点图展示转录本长度与覆盖率的关系。其中，x轴表示转录本的长度，y轴表示重叠群对转录本的覆盖率，颜色从黑色到灰色表示组装长度从短到长。图7中的结果表明，大部分转录本都可以组装出全长。
图8示出了样品1组装的覆盖长度分布，其中使用直方图展示组装的重叠群对转录本覆盖长度的分布。其中，x轴表示组装的重叠群对转录本的覆盖长度，y轴表示频数。图8中的结果表明，重叠群组装出全长的频数较高。

Claims (22)

  1. 一种通过单链滚环扩增获得双链序列的方法，所述方法包括：
    1)以第一引物对单链环状DNA进行滚环扩增反应，获得扩增序列，所述第一引物与所述单链环状DNA部分区域互补，所述单链环状DNA具有可致所述单链环状DNA开环的断开机制；
    2)通过所述断开机制使所述单链环状DNA开环，获得单链线性DNA；
    3)以所述单链线性DNA为第二引物、以1)中获得的扩增序列为模板进行扩增反应，获得扩增的双链序列。
  2. 根据权利要求1所述的方法，所述单链环状DNA来自对DNA样本或cDNA样本进行环化，通过PCR或接头连接，在所述单链环状DNA中引入特殊碱基或特异性序列。
  3. 根据权利要求1或2所述的方法，所述第一引物为DNA引物或RNA引物。
  4. 根据权利要求1-3任一项所述的方法，所述断开机制为所述单链环状DNA包含特定区域，所述特定区域在生化反应下发生断裂，从而导致所述单链环状DNA开环。
  5. 根据权利要求4所述的方法，所述特定区域包括特殊碱基和/或特异性序列。
  6. 根据权利要求5所述的方法，所述特殊碱基为次黄嘌呤脱氧核核苷、脱氧尿嘧啶核苷酸、RNA碱基、AP位点或甲基化位点。
  7. 根据权利要求6所述的方法，所述特殊碱基为次黄嘌呤脱氧核核苷，使用核酸内切酶V进行消化切除，从而使单链环状DNA开环。
  8. 根据权利要求6所述的方法，所述特殊碱基为脱氧尿嘧啶核苷酸，使用UDG酶或APE1酶进行识别和切除，从而使单链环状DNA开环。
  9. 根据权利要求6所述的方法，所述特殊碱基为RNA碱基，使用RNaseA或RNaseH进行识别和切除，从而使单链环状DNA开环。
  10. 根据权利要求6所述的方法，所述特殊碱基为AP位点，使用APE1酶进行识别和切除，从而使单链环状DNA开环。
  11. 根据权利要求6所述的方法，所述特殊碱基为甲基化C碱基位点，使用APOBEC脱氨酶、TET2酶或亚硫酸氢钠进行处理，将甲基化C碱基转化为dU碱基，再通过UDG酶或APE1酶进行识别和切除，从而使单链环状DNA开环。
  12. 根据权利要求5所述的方法，所述特异性序列为限制性内切酶识别位点或蛋白质的特异性结合位点。
  13. 根据权利要求12所述的方法，所述限制性内切酶识别位点为富含AT碱基序列的区域；优选地，所述限制性内切酶识别位点为Chlamydomonas内切酶识别位点或Neurospora crassa内切酶识别位点。
  14. 根据权利要求12所述的方法，所述蛋白质的特异性结合位点为CRISPR/Cas基因编辑系统中向导RNA的识别区域；优选地，为CRISPR/Cas9中向导RNA的识别区域。
  15. 根据权利要求1-14任一项所述的方法，在对单链环状DNA进行滚环扩增反应过程中或之后加入单链DNA结合蛋白、焦磷酸酶和TE缓冲液。
  16. 根据权利要求1-14任一项所述的方法，在对单链环状DNA进行滚环扩增反应过程中或之后加入解旋酶。
  17. 根据权利要求16所述的方法，所述解旋酶为具有3’到5’方向解旋能力的A型解旋酶，优选为Rep解旋酶、UvrD解旋酶、Heli308解旋酶、PcrA解旋酶、或RecD2解旋酶。
  18. 一种核酸测序文库构建方法，所述方法包括：
    1)通过权利要求1-17任一项所述的方法获得扩增的双链序列；
    2)将所述双链序列进行测序文库构建，获得所述核酸测序文库。
  19. 根据权利要求18所述的方法，使用LFR技术(长片段读取技术)进行核酸测序文库构建，获得所述核酸测序文库。
  20. 根据权利要求18或19所述的方法，所述核酸测序文库是mRNA全长转录本文库。
  21. 一种测序方法，所述方法包括：
    1)通过权利要求1-17任一项所述的方法获得扩增的双链序列，或者权利要求18-20任一项所述的方法获得核酸测序文库；
    2)对所述扩增的双链序列或核酸测序文库进行测序。
  22. 根据权利要求21所述的测序方法，所述测序为高通量测序，例如第二代测序或第三代测序。
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