DNA recombination method based on RAA

Abstract

The invention belongs to the technical field of biology, and particularly relates to a DNA recombination method based on RAA, which comprises the following steps: carrying out RAA on a to-be-recombined gene segment through an RAA primer, in the RAA process, generating a gene segment product, the RAA primer comprises a connection primer, the connection primer has a first part and a second part which are homologous with the two to-be-recombined gene segments respectively, and hybridizing the gene fragment products on the homologous sites, and then combining and prolonging the hybridized fragments by using Bsu DNA polymerase to complete the fusion of the gene fragments hybridized on the homologous sites. The invention provides a DNA (deoxyribonucleic acid) recombination method based on RAA (recombinase of amplification), which can be used for quickly, simply, conveniently and accurately fusing DNA fragments and can be used for realizing insertion, deletion and base mutation of the DNA fragments.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant DNA method based on RAA. Background Art

[0002] Recombinant DNA technology is the technology of assembling DNA fusion fragments, which constitutes the foundation of genetic engineering. DNA fusion fragments are not only widely used in genetic engineering fields including recombinant protein expression, transgenic product development and mutant construction, but also in the field of pathogen detection. So far, a variety of traditional DNA recombinant technologies can be used to construct DNA fusion fragments, but these methods usually require the construction of recombinant plasmids through molecular cloning technology, and then produce DNA fusion fragments under the action of restriction endonucleases. Therefore, they are not only time-consuming and labor-intensive, but also easy to introduce unnecessary sequences.

[0003] RAA is a new isothermal nucleic acid amplification technology that has attracted much attention due to its constant temperature, sensitivity, specificity, rapidity, and ease of use. The RAA reaction is to amplify the target gene under isothermal conditions (37°C) by designing a pair of specific primers for the target gene and using DNA recombinase, polymerase, and single-stranded DNA binding protein. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of existing recombinant DNA technology and to provide a recombinant DNA method based on RAA.

[0005] The technical solution adopted by the present invention is as follows: a recombinant DNA method based on RAA, comprising performing RAA on a gene fragment to be recombined through an RAA primer, during the RAA process, a gene fragment product is generated, the RAA primer comprises a connecting primer, the connecting primer has a first part and a second part which are homologous to two gene fragments to be recombined, respectively, so that the gene fragment product hybridizes at the homologous site, and then the Bsu DNA polymerase combines with the hybridized fragment and extends it, thereby completing the fusion of the gene fragments hybridized at the homologous site.

[0006] The method is used for fusing fragment A and fragment B, and the method comprises the following steps:

[0007] (1) designing primers FP1 and RP1 according to the sequence of fragment A, and designing primers FP2 and RP2 according to the sequence of fragment B, wherein RP1 is a connecting primer, and RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment B;

[0008] (2) Primers FP1 and RP1, as well as primers FP2 and RP2, were used to perform RAA on gene fragment A and gene fragment B, respectively. Product As was produced during the RAA process of fragment A, and product Bs was produced during the RAA process of fragment B. As and Bs fragments hybridized at homologous sites, and Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the fusion of fragments A and B.

[0009] The method is used to insert fragment C between fragment A and fragment B, and the method comprises the following steps:

[0010] (1) Designing primers FP1 and RP1 based on the sequence of fragment A, designing primers FP2 and RP2 based on the sequence of fragment B, and designing primers FP3 and RP3 based on the sequence of fragment C, wherein RP1 and RP3 are connecting primers;

[0011] RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment C;

[0012] RP3 has two parts: the first part is a sequence homologous to fragment C, and the second part is a sequence homologous to fragment B;

[0013] (2) Primers FP1 and RP1, primers FP2 and RP2, and primers FP3 and RP3 were used to perform RAA on gene fragments A, gene fragment B, and gene fragment C. During the RAA process of fragments A, fragment B, and fragment C, products As, product Bs, and product Cs were generated. The two ends of the generated Cs hybridized with the As and Bs fragments at the homologous sites, respectively. Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the sequential fusion of fragments A, C, and B.

[0014] The method is used to delete fragment B in a gene composed of fragments A, B, and C connected in sequence, to obtain a fusion DNA directly connected between fragments A and C, and the method comprises the following steps:

[0015] (1) Design primers FP1 and RP1 based on the sequence of fragment A, and design primers FP2 and RP2 based on the sequence of fragment C, wherein RP1 is a connecting primer;

[0016] RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment C;

[0017] (2) Using primers FP1 and RP1, and primers FP2 and RP2, RAA is performed on the gene composed of fragments A, B, and C in sequence, producing products As and Cs. The As and Cs fragments hybridize at homologous sites, and Bsu DNA polymerase binds to the hybridized fragments and extends them.

[0018] The method is used to construct a fusion DNA with base mutations, wherein the fusion DNA includes a directly connected fragment A and a fragment B, and the fragment B is close to a partial base mutation of the fragment A. The method comprises the following steps:

[0019] (1) Designing primers FP1 and RP1 based on the sequence of fragment A, and designing primers FP2 and RP2 based on the sequence of fragment B, wherein RP1 is a connecting primer;

[0020] RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment B, and a mutated base is constructed in the sequence homologous to fragment B;

[0021] The bases to be mutated are constructed in the FP2 sequence;

[0022] (2) Fragments A and B were subjected to RAA using primers FP1 and RP1, and primers FP2 and RP2, to produce products As and Bs. Fragments As and Bs hybridized at homologous sites, and Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the fusion of fragments A and B with mutation sites.

[0023] The connecting primer consists of a first part and a second part, the length of the first part is 30, and the length of the second part is 15.

[0024] The RAA primers include inner primers and outer primers. The inner primers are designed according to the inner end sequence of the gene fragment to be fused for connection, and the outer primers are designed according to the outer end sequence of the gene fragment to be fused that is not used for connection. The usage ratio of the outer primers to the inner primers is 2:1.

[0025] The present invention provides a RAA-based recombinant DNA method, which can quickly, simply and accurately fuse DNA fragments, and can achieve DNA fragment insertion, deletion and base mutation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0027] Figure 1 .Schematic diagram of the RAA fusion process;

[0028] Figure 2 . Electrophoresis analysis of three groups of RAA products;

[0029] Figure 3. Optimization of primer conditions. (a) Optimization of the length of the connecting primer (RP1 = HSA + HSB), (b) Optimization of the ratio of the amount of the outer and inner primers;

[0030] Figure 4 . Schematic diagram of the principle of fusion RAA for DNA fragment insertion, deletion and base mutation;

[0031] Figure 5 .DNA sequence modification. (a) Experimental results of RAA fusion for DNA sequence insertion, (b) Experimental results of RAA fusion for DNA fragment deletion, (c) Results of wild-type and mutant primer sequences and wild-type and mutant product size comparison;

[0032] Figure 6 .Sequence alignment results of base mutation scheme. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0034] The fusion RAA reactions in the following examples were all carried out using the RAA kit of Jiangsu Qitian Gene Biotechnology Co., Ltd., which included enzyme components, specifically recombinase, single-strand binding protein (SSB) and DNA polymerase. Recombinase is one of the core components, which is responsible for recognizing the specific sequence of double-stranded DNA and initiating the reaction; single-strand binding protein can stabilize single-stranded DNA to prevent it from re-forming double strands, ensuring the smooth progress of the reaction; DNA polymerase is used to extend the DNA chain and synthesize a new DNA chain based on the template chain.

[0035] The following examples

[0036] Embodiment 1:

[0037] In order to reduce the cost of the test without reducing the performance of the test, the lyophilized powder in each test tube was dissolved and divided into two parts. The specific operation was as follows: 25 μL of buffer V and a certain volume of purified water were added to the test tube to dissolve the particles in the tube, and after mixing, the solution was equally divided into two new reaction tubes. The final concentration of the outer primer (400nM) was twice the final concentration of the inner primer (200nM), so each reaction system included 2μL of 10μM outer primers FP1 and RP2, 1μL of 10μM inner primers FP2 and RP1, and 2.5μL of magnesium acetate I and a certain volume of template were added to form a total reaction system of 25μL. It is worth noting that magnesium acetate I should be added to the reaction tube cap first, and then the tube should be quickly inverted to ensure that the reactions start at the same time. Unless otherwise specified, each reaction was incubated at 37°C for 40 minutes.

[0038] like Figure 1As shown, firstly, fragment A and fragment B are amplified according to the RAA principle. In the RAA process of fragment A, product As (dsDNA and 5'-3'ssDNA) is produced, while in the RAA process of fragment B, product Bs (3'-5'ssDNA and dsDNA) is produced. The produced As and Bs fragments hybridize at homologous sites. Then, Bsu DNA polymerase binds to the hybridized fragments and extends them, completing the fusion of fragments A and B.

[0039] The recombinase-primer complex initiates RAA by scanning the double-stranded DNA template to find the homologous sequence of the primer, and then the complex invades the homologous sequence to form a D-loop ring structure, in which the displaced ssDNA is stabilized by the ssDNA binding protein. Once the recombinase dissociates, the 3' end of the primer binds to the DNA polymerase and is extended. Since ssDNA is formed during the RAA process, the ssDNA can be used to introduce overlapping sequences to assemble DNA fragments.

[0040] In this embodiment, fragment A is a portion of the nuc gene sequence of methicillin-resistant Staphylococcus aureus (single straight line underline, Table 2) and fragment B is a portion of the mecA gene sequence of methicillin-resistant Staphylococcus aureus (wavy line underline, Table 2). In order to fuse DNA fragments A and B, a pair of RAA primers FP1 and RP1, FP2 and RP2 are designed according to fragments A and B, respectively, wherein RP1 must contain a sequence homologous to FP2 (red line). In this embodiment, three sets of primers are designed (as shown in Table 1), which can generate fusion DNA fragments of 449 bp, 385 bp and 420 bp, respectively (Table 2). Agarose gel results show that the three sets of primer combinations can produce fusion product bands of the expected size ( Figure 2 ). We further purified these DNA bands for Sanger sequencing and observed that the product sequences were completely correct, proving the feasibility of RAA to assemble DNA fragments. We named this DNA fusion method "fusion RAA".

[0041] Table 1. Three sets of RAA fusion primer sequences

[0042]

[0043] Table 2. Sequences of three sets of DNA fusion products. (Fragment A is underlined with a single straight line, and fragment B is underlined with a wavy line)

[0044]

[0045]

[0046] Example 2 Effect of primer sequence length:

[0047] The linker primer RP1 of the fusion RAA reaction consists of two parts: one part is a sequence homologous to the A segment (HSA), and the other part is a sequence homologous to the B segment (HSB), that is, RP1 = HSA + HSB. The length of the HSA and HSB sequences has an important influence on the occurrence of fusion.

[0048] In this example, the fragment A sequence is a part of the nuc gene sequence of methicillin-resistant Staphylococcus aureus (Genbank ID: EF529596.1, positions 157-385), and the fragment B sequence is a part of the mecA gene sequence of methicillin-resistant Staphylococcus aureus (Genbank ID: MH798869.1, positions 167-389). In this example, three HSAs (20 bp, 25 bp, and 30 bp) and three HSBs (15 bp, 20 bp, and 25 bp), i.e., nine RP1 sequences (Table 3), were selected and fused with RAA reactions to explore the optimal sequence length combination of RP1. The steps of the fusion RAA reaction are the same as those in Example 1.

[0049] The results showed that the best values ​​were HSA 30 and HSB 15 ( Figure 3 a).

[0050] Table 3. Primer sequences

[0051]

[0052] Influence of the ratio of external and internal primers in Example 3:

[0053] Since the outer primers ( Figure 1 FP1 and RP2 in the primers are the main primers for generating fusion fragments, so the outer primers and inner primers ( Figure 1 The amount of RP1 and FP2 in the reaction will affect the fusion effect. We used four external and internal primer ratios (1:1, 2:1, 3:1 and 4:1) to perform the fusion RAA reaction and explore the optimal external and internal primer ratio. The steps of the fusion RAA reaction are the same as those in Example 1. In this example, the fragment A sequence is a part of the nuc gene sequence of methicillin-resistant Staphylococcus aureus (Genbank ID: EF529596.1, positions 157-385), and the fragment B sequence is a part of the mecA gene sequence of methicillin-resistant Staphylococcus aureus (Genbank ID: MH798869.1, positions 167-389). The primer sequences used are the same as FP1, RP1 (30-15), FP2 and RP2 in Table 3.

[0054] The results showed that the optimal ratio of external and internal primers was 2:1. Figure 3 b).

[0055] Example 4: Application of RAA fusion to fragment insertion:

[0056] The general steps of this embodiment are the same as those of Example 1, and the total volume of each fusion RAA is 50 μL, including 25 μL buffer V, 10 μL purified water, four inner primers (1 μL of each primer, 10 μM) and two outer primers (2 μL of each primer, 10 μM), 5 μL magnesium acetate I and 2 μL MRSA (methicillin-resistant Staphylococcus aureus) DNA. Finally, the solution was mixed thoroughly and incubated at 37° C. for 40 minutes. The amplified product was purified using a PCR purification kit and then analyzed by 2% gel electrophoresis.

[0057] This example attempts to insert a 225 bp long fragment (fragment C, wavy line underline) between fragment A (173 bp, single straight line underline) and fragment B (198 bp, double straight line underline) (Table 5). To this end, this example designs three pairs of primers (Table 4). RP1 and RP3 carry sequences homologous to the 5' end of primers FP3 and FP2, respectively ( Figure 4 a). Gel electrophoresis of the fusion RAA reaction showed a clear band of 596 bp, which is the size of the ACB fusion product ( Figure 5 This result not only confirmed the effectiveness of fusion RAA in the precise insertion of DNA fragments, but also demonstrated its ability to assemble three DNA fragments.

[0058] Table 4. Primer sequences for DNA fragment insertion protocol

[0059]

[0060] Table 5. Sequences of ACB fusion products. (Fragment A is underlined with a single straight line, fragment C is underlined with a wavy line, and fragment B is underlined with a double straight line)

[0061]

[0062] Example 5: Application of RAA fusion to fragment deletion:

[0063] The general steps of this example are the same as those of Example 1, and the total volume of each fusion RAA is 50 μL, including 25 μL buffer V, 10 μL purified water, 4 μL outer primer (10 μM), 2 μL inner primer (10 μM), 5 μL magnesium acetate I and 2 μL MRSA DNA. Finally, the solution was thoroughly mixed and incubated at 37° C. for 40 minutes. The amplified product was purified with a PCR purification kit and then analyzed by 2% gel electrophoresis.

[0064] In this example, fragment ABC is a continuous sequence in the mecA gene sequence of methicillin-resistant Staphylococcus aureus (Genbank ID: KC243783.1, positions 158-1650), and the feasibility of fusing RAA for DNA fragment deletion was explored ( Figure 4 b). To this end, four sets of fusion RAA primer pairs targeting different genomic sites of methicillin-resistant Staphylococcus aureus were designed (Table 6), of which RP1 is a connecting primer with a sequence homologous to primer FP2, in order to generate DNA fragments with different lengths (27 bp, 247 bp, 694 bp and 1118 bp) and sequence deletions (353 bp, 378 bp, 346 bp and 375 bp) (Table 7). The electrophoresis results showed that the expected size of the fusion product ( Figure 5 b), indicating that DNA fragments can be deleted by fusion with RAA.

[0065] Table 6. Primer sequences for DNA sequence fragment deletion scheme

[0066]

[0067] Table 7. Product sequences of DNA sequence fragment deletions. (Fragment A is underlined with a single straight line, and fragment C is underlined with a wavy line)

[0068]

[0069]

[0070] Example 6: Fusion of RAA is used to introduce base mutations:

[0071] The general steps of this example are the same as those of Example 1, and the total volume of each fusion RAA is 50 μL, including 25 μL buffer V, 10 μL purified water, 4 μL outer primer (10 μM), 2 μL inner primer (10 μM), 5 μL magnesium acetate I and 2 μL MRSA DNA. Finally, the solution was thoroughly mixed and incubated at 37° C. for 40 minutes. The amplified product was purified with a PCR purification kit and then analyzed by 2% gel electrophoresis.

[0072] In this example, fragment A is a portion of the nuc gene sequence of methicillin-resistant Staphylococcus aureus (single straight line underline, Table 9), and fragment B is a portion of the mecA gene sequence of methicillin-resistant Staphylococcus aureus (wavy line underline, Table 9). The feasibility of fusion RAA for introducing base mutations was explored ( Figure 4c). To this end, as shown in Table 8, two sets of primers were designed, including mutant primers (mut) and wild-type primers (wt). MutFP2 and mutRP1 were provided with mutant bases. MutRP1 was used as a connecting primer, on which a sequence homologous to primer mutFP2 was provided. The wild-type primer (wt) was used as a control. Since the wild-type and mutant products were of the same size ( Figure 5 c, Table 9), and the precise occurrence of the point mutation was verified by Sanger sequencing analysis of the product ( Figure 6 ).

[0073] Table 8. Primer sequences for DNA sequence base mutation scheme

[0074]

[0075] Table 9. Product sequences of DNA sequence base mutations. (Fragment A is a single straight line underline, and fragment B is a wavy line underline)

[0076]

[0077] In summary, RAA fusion can quickly, easily and accurately fuse DNA fragments, and can achieve DNA fragment insertion, deletion and base mutation.

[0078] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. A recombinant DNA method based on RAA, characterized in that: The method comprises performing RAA on the gene fragments to be recombined by using RAA primers. During the RAA process, gene fragment products are generated. The RAA primers include connecting primers. The connecting primers have a first part and a second part which are respectively homologous to two gene fragments to be recombined, so that the gene fragment products hybridize at homologous sites. Then, Bsu DNA polymerase binds and extends the hybridized fragments, thereby completing the fusion of the gene fragments hybridized at the homologous sites.

2. The RAA-based recombinant DNA method according to claim 1, characterized in that: The method is used for fusing fragment A and fragment B, and the method comprises the following steps: (1) designing primers FP1 and RP1 according to the sequence of fragment A, and designing primers FP2 and RP2 according to the sequence of fragment B, wherein RP1 is a connecting primer, and RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment B; (2) Primers FP1 and RP1, as well as primers FP2 and RP2, were used to simultaneously perform RAA on gene fragments A and B. Product As was produced during the RAA process of fragment A, and product Bs was produced during the RAA process of fragment B. The As and Bs fragments hybridized at homologous sites, and Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the fusion of fragments A and B.

3. The RAA-based recombinant DNA method according to claim 1, characterized in that: The method is used to insert fragment C between fragment A and fragment B, and the method comprises the following steps: (1) Designing primers FP1 and RP1 based on the sequence of fragment A, designing primers FP2 and RP2 based on the sequence of fragment B, and designing primers FP3 and RP3 based on the sequence of fragment C, wherein RP1 and RP3 are connecting primers; RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment C; RP3 has two parts: the first part is a sequence homologous to fragment C, and the second part is a sequence homologous to fragment B; (2) Primers FP1 and RP1, primers FP2 and RP2, and primers FP3 and RP3 were used to perform RAA on gene fragments A, gene fragment B, and gene fragment C. During the RAA process of fragments A, fragment B, and fragment C, products As, product Bs, and product Cs were generated. The two ends of the generated Cs hybridized with the As and Bs fragments at the homologous sites, respectively. Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the sequential fusion of fragments A, C, and B.

4. The RAA-based recombinant DNA method according to claim 1, characterized in that: The method is used to delete fragment B in a gene composed of fragments A, B, and C connected in sequence, to obtain a fusion DNA directly connected between fragments A and C, and the method comprises the following steps: (1) Design primers FP1 and RP1 based on the sequence of fragment A, and design primers FP2 and RP2 based on the sequence of fragment C, wherein RP1 is a connecting primer; RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment C; (2) Primers FP1 and RP1, as well as primers FP2 and RP2, were used to perform RAA on the gene consisting of fragments A, B, and C in sequence, producing products As and Cs. The As and Cs fragments hybridized at homologous sites, and Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the fusion of fragments A and C.

5. The RAA-based recombinant DNA method according to claim 1, characterized in that: The method is used to construct a fusion DNA with base mutations, wherein the fusion DNA includes a directly connected fragment A and a fragment B, and the fragment B is close to a partial base mutation of the fragment A. The method comprises the following steps: (1) Designing primers FP1 and RP1 based on the sequence of fragment A, and designing primers FP2 and RP2 based on the sequence of fragment B, wherein RP1 is a connecting primer; RP1 has two parts: the first part is a sequence homologous to fragment A, and the second part is a sequence homologous to fragment B, and a mutated base is constructed in the sequence homologous to fragment B; The bases to be mutated are constructed in the FP2 sequence; (2) Fragments A and B were subjected to RAA using primers FP1 and RP1, and primers FP2 and RP2, to produce products As and Bs. Fragments As and Bs hybridized at homologous sites, and Bsu DNA polymerase bound to the hybridized fragments and extended them, completing the fusion of fragments A and B with mutation sites.

6. The RAA-based recombinant DNA method according to any one of claims 1 to 5, characterized in that: The connecting primer consists of a first part and a second part, the length of the first part is 30, and the length of the second part is 15.

7. The RAA-based recombinant DNA method according to any one of claims 1 to 5, characterized in that: The RAA primers include inner primers and outer primers. The inner primers are designed according to the inner end sequence of the gene fragment to be fused for connection, and the outer primers are designed according to the outer end sequence of the gene fragment to be fused that is not used for connection. The usage ratio of the outer primers to the inner primers is 2:1.

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