CN117802090A - Rapid synthesis method of long fragment gene - Google Patents
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Abstract
The invention discloses a rapid synthesis method of a long fragment gene. The method comprises the following steps: designing and synthesizing primers in groups, wherein each group comprises 2 pairs (4) of primers; grouping and sectionally assembling the synthesized primers by using an overlay PCR method, and simultaneously carrying out multiple reactions; and (5) purifying and sequencing the final PCR product. The invention adopts a grouping and sectioning mode to carry out gene amplification, thereby not only avoiding the problem of low success rate of the long fragment amplified by the traditional overlay PCR, but also saving the synthesis time, having the advantages of low cost, easy realization and easy popularization without additional reagents, and having good application prospect in the technical field of gene synthesis.
Description
Technical Field
The invention belongs to the technical field of gene synthesis, and particularly relates to a rapid synthesis method of a long fragment gene.
Background
DNA is a main carrier of genetic information, is a life information base, and DNA synthesis is an important tool for exploring life mysterious. The technology of synthesizing double-stranded DNA molecule in vitro is gene synthesis. Gene synthesis typically employs chemical methods to stepwise synthesize a short DNA sequence into a complete long-chain DNA molecule. At present, the domestic gene synthesis is mainly based on the traditional first generation column synthesis technology, firstly, sequences of 60-120nt are synthesized through a column synthesis method, and then, the sequences are amplified and spliced into genes of about 1 kb. However, the conventional overlay PCR amplified long fragments have a problem of low success rate.
The conventional synthesis cycle of each enterprise in the market at present, the 1kb gene generally needs 6-10 days, and the 8kb gene needs 20-25 days. The longer the gene, the longer the time consuming. Enterprise synthesis has the problems of long period and high cost. Therefore, there is a need to develop a low-time-consuming and low-cost gene synthesis method to better meet the demands of scientific research and the like.
Disclosure of Invention
The invention aims to provide a long fragment gene synthesis method with low time consumption and low cost.
In order to achieve the above object, the present invention provides a method for synthesizing a long fragment gene.
The synthesis method of the long fragment gene provided by the invention comprises the following steps:
1) Dividing a target long fragment gene sequence into m sections, and then designing an overlay PCR primer for each section to obtain m groups of primers; each group of primers comprises 2 forward primers and 2 reverse primers, the 2 forward primers are respectively marked as a primer F and a primer F-T, the 2 reverse primers are respectively marked as a primer R and a primer R-T, wherein the primer F-T is a reverse complementary sequence of the primer R-T, the two primers are both used as templates for the overlay PCR amplification, an overlapping section is arranged between the 2 forward primers, an overlapping section is arranged between the 2 reverse primers, and an overlapping section is arranged between two adjacent groups of primers;
2) Respectively carrying out the 1 st round of over lap PCR amplification on each group of primers at the same time to obtain m over lap PCR amplification products; then, mixing two adjacent sets of the overlay PCR amplification products, and simultaneously carrying out 2 nd round of overlay PCR amplification to synthesize small fragments in the two adjacent sets of the overlay PCR amplification products into large fragments; and by analogy, obtaining a final overlay PCR amplification product by multiple rounds of overlay PCR amplification, namely the long fragment gene.
In the above method, in the step 1), the length of the effective extension fragment of each primer set (i.e., the amplified product obtained after the overlay PCR amplification using each primer in each primer set) may be 110 to 120.
In the above method, in the step 1), since the primer synthesis time of less than 60bp is shortest and the cost is optimal when the primers are synthesized commercially, the length of each primer in each group of primers is less than or equal to 59bp.
In the above method, in the step 1), the over lap PCR primer can be designed according to a conventional design method and principle using a primer design software commonly used in the art, and the GC content is to be paid attention to the design of the primer so that the portion has a proper Tm value, and typically, the Tm value of the over lap PCR primer is more than 55 ℃.
In one embodiment, the snap primer design software may be used to design the overlay PCR primer.
In the above method, in the step 1), the length of the overlapping region may be 20 to 25bp.
In the above method, in the step 1), m segments may be respectively designated as segment 1, segment 2, segment 3, … …, and so on, segment m-1, segment m, and primers designed for segment 1 are designated as group 1 primers (while F, F-T, R and R-T in group 1 primers are designated as F1, F-T1, R1 and R-T1, respectively), primers designed for segment 2 are designated as group 2 primers (while F, F-T, R and R-T in group 2 primers are designated as F2, F-T2, R2 and R-T2, respectively), … …, and so on, primers designed for segment m-1 are designated as group 1 primers (while F, F-95 and R-T in group 1 are designated as F (m-1), F-T (m-1), R (m-1) and R (m-1) are designated as F-1, R-T) and Tm) respectively), primers designed for segment m-1 are designated as group 2 primers (while F, F-T, R and R-T in group 2 are designated as F2, F-T2, R2 and R-T2, and Tm as m-1, respectively.
The meaning of the overlapping section between the two adjacent sets of primers means that the overlapping section is arranged between the primer R1 in the 1 st set of primers and the primer F2 in the 2 nd set of primers, the overlapping section is arranged between the primer R2 in the 2 nd set of primers and the primer F3 in the 3 rd set of primers, … …, and the like, and the overlapping section is arranged between the primer R (m-1) in the m-1 th set of primers and the primer Fm in the m-th set of primers.
In the above method, in the step 2), the product of the previous round of the overlay PCR amplification is directly used as a template for the next round of the overlay PCR amplification without DNA recovery and purification.
In the above method, in the step 2), the final Overlap PCR amplification product has a length of (2 n-1 X 115) bp, wherein n is the number of amplification rounds. If n is 4, 4 rounds of overlay PCR amplification are carried out, and finally a fragment with the length of about 920bp can be obtained; and 5 rounds of overlay PCR amplification are carried out when n is 5, and finally fragments with the length of about 1800bp can be obtained.
In the above method, in the above step 2), the above-mentioned over-lap PCR amplified product obtained by amplifying the above-mentioned 1 st primer set is referred to as over-lap PCR amplified product 1, the above-mentioned over-lap PCR amplified product obtained by amplifying the above-mentioned 2 nd primer set is referred to as over-lap PCR amplified product 2, … …, and so on, the above-mentioned over-lap PCR amplified product obtained by amplifying the above-mentioned m-1 st primer set is referred to as over-lap PCR amplified product m-1, and the above-mentioned over-lap PCR amplified product obtained by amplifying the above-mentioned m-th primer set is referred to as over-lap PCR amplified product m.
The meaning of two adjacent sets of the overlay PCR amplification products being mixed is that the overlay PCR amplification product 1 and the overlay PCR amplification product 2 are mixed, the overlay PCR amplification product 3 and the overlay PCR amplification product 4 are mixed, … …, and the overlay PCR amplification product m-1 and the overlay PCR amplification product m are mixed. Similarly, after the other round of overlay PCR amplification, two adjacent sets of overlay PCR amplification products were mixed in pairs according to the same method.
If the situation that 1 over-lap PCR amplification product exists after two adjacent two groups of over-lap PCR amplification products are mixed, the over-lap PCR amplification products do not need to participate in the over-lap PCR amplification of the round, and directly participate in the next round of over-lap PCR amplification.
The segmented and grouped gene amplification mode adopted by the invention can obviously reduce the mismatch probability and improve the probability of correct extension.
The method of step 2) above further comprises the step of cloning the final overlay PCR amplification product into an expression vector and sequencing the resulting recombinant vector.
In one embodiment of the present invention, the method of cloning the final overlay PCR amplification product into an expression vector and sequencing the resulting recombinant vector may comprise the steps of:
a. recovering and purifying the final overlay PCR amplification product to obtain a purified final overlay PCR amplification product;
b. cloning the purified final Overlap PCR amplification product into an expression vector to obtain a recombinant vector;
c. transforming the recombinant vector into escherichia coli, culturing overnight, selecting a monoclonal colony, and carrying out colony PCR identification to obtain colony PCR positive clones;
d. and (3) amplifying and culturing the colony PCR positive clone, extracting and purifying plasmids, and verifying by adopting a gene sequencing technology to complete gene synthesis.
Wherein, in step a, the final Overlap PCR amplification product may be purified in any manner known in the art. In a specific embodiment of the invention, the final overlay PCR amplification product is purified using silica gel column purification techniques.
In step b, the expression vector may be any expression vector known in the art. In a specific embodiment of the invention, the expression vector is a pEG BacMam vector.
In the step c, the colony PCR identification comprises the step of preliminary verification of the sequences obtained by PCR amplification.
In step d, the purpose of the verification using gene sequencing techniques is to ensure that the gene is free of any form of mutation (e.g., single base changes or insertions or deletions, small fragment changes or insertions or deletions, etc.).
In the above method, the long fragment gene may be any gene having a length of more than 200bp, preferably a gene having a length of more than 1000 bp.
In a specific embodiment of the invention, the long fragment gene is the SLC22A2 gene with the length of 1665bp, and the specific nucleotide sequence is shown in sequence 1. The invention designs 15 groups of primers aiming at long fragment gene SLC22A2, and the sequence of each group of primers is shown in the following table 1.
In order to achieve the above purpose, the invention also provides a new application of the method.
The invention provides the application of the method in improving the synthesis efficiency of the long fragment gene and/or shortening the synthesis time of the long fragment gene and/or reducing the synthesis cost of the long fragment gene.
The invention provides a synthesis method of a long fragment gene, which is improved on the basis of the traditional overlay PCR, optimizes a fragment connection method, creatively utilizes a grouping and sectioning mode to carry out gene amplification, carries out multiple reactions respectively and simultaneously, saves more time and has more obvious advantages as the gene fragment is longer.
Compared with the prior art, the invention has the following beneficial effects: the long fragment gene synthesis method provided by the invention effectively avoids great assembly complexity caused by multiple oligonucleotide types in a sample cell, improves the synthesis efficiency, shortens the synthesis time, uses common reagents for gene amplification, does not need additional reagents, and has the advantages of simple operation, low cost, easy realization and easy popularization.
Drawings
FIG. 1 is a simplified operational flow diagram of one embodiment of the present invention.
FIG. 2 is a diagram and a schematic diagram of an example of primer design in the present invention. A is an example graph; b is a schematic diagram.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The amplification reagents and gel recovery kit used in the following examples were all manufactured by Nanjinopran Biotechnology Co., ltd.
Example 1, rapid Synthesis of Long fragment Gene
The flow of the rapid synthesis method of the long fragment gene of the present invention is shown in FIG. 1.
1. Primer design and synthesis
Dividing the target long fragment gene sequence into m segments, marking the m segments as segment 1, segment 2, segment 3, … …, and so on, segment m-1, segment m respectively, and then designing the overlay PCR primers by Snapgene primer design software for each segment respectively, marking the primers designed for segment 1 as group 1 primers, marking the primers designed for segment 2 as group 2 primers, marking the primers designed for segment 3 as group 3 primers, … …, and so on, marking the primers designed for segment m-1 as group m-1 primers, marking the primers designed for segment m as group m primers, and obtaining m groups of primers. A schematic of the primer design for each set is shown in FIG. 2.
Each set of primers contained 2 pairs (4) of primers, 2 of which were forward primers (F, F-T, where T is an abbreviation for template) and 2 were reverse primers (R, R-T). Wherein, overlap sections are arranged between 2 forward primers, overlap sections are arranged between 2 reverse primers, and overlap sections are arranged between two adjacent groups of primers. The primer F-T and the primer R-T both play a role of a template, and the primer F-T is a reverse complementary sequence of the primer R-T. The effective extension fragment length of each primer set is 110-120bp. Because the synthesis time of the primer smaller than 60bp is shortest and the cost is optimal in the existing commercial synthesis, the length of each primer in each group of primers is less than or equal to 59bp. All overlapping segments are 20-25bp in length. The Tm values of all the designed primers are generally > 55 ℃.
For example, the number of the cells to be processed,
the 2 forward primers in the 1 st set of primers were designated as F1 and F-T1, the 2 reverse primers were designated as R1 and R-T1, the 2 forward primers in the 2 nd set of primers were designated as F2 and F-T2, the 2 reverse primers were designated as R2 and R-T2, … …, and the 2 forward primers in the m-1 st set were designated as F (m-1) and F-T (m-1), the 2 reverse primers were designated as R (m-1) and R-T (m-1), the 2 forward primers in the m-th set were designated as Fm and F-Tm, and the 2 reverse primers were designated as Rm and R-Tm.
For the 1 st set of primers, an overlap region is provided between primer F1 and primer F-T1, between primer R1 and primer R-T1, and between primer R1 and primer F2.
For the 2 nd set of primers, an overlap region is provided between primer F2 and primer F-T2, between primer R2 and primer R-T2, and between primer R2 and primer F3.
… …, in a subspecies of the push,
for the m-1 th set of primers, an overlap region is provided between primer F (m-1) and primer F-T (m-1), between primer R (m-1) and primer R-T (m-1), and between primer R (m-1) and primer Fm.
15 sets of primers were designed in total according to the above method, taking the long fragment gene SLC22A2 with length of 1665bp as an example, and the sequences of each set of primers are shown in Table 1, wherein the primers numbered F1, R1, F-T1 and R-T1 are the 1 st set of primers, the primers numbered F2, R2, F-T2 and R-T2 are the 2 nd set of primers, … …, and the primers numbered F15, R15, F-T15 and R-T15 are the 15 th set of primers. The primer sequences in Table 1 were synthesized by Beijing qing Biotech Co., ltd.
TABLE 1
2. Grouping and segmented gene amplification
The primer groups synthesized in the first step of grouping and sectional assembly are subjected to multiple reactions respectively and simultaneously, and each round of PCR reaction is performed, two adjacent primer groups are selected for amplification, namely two fragments are connected each time, so that the mismatch probability can be remarkably reduced, and the probability of correct extension is improved. The PCR product of the previous round is directly used as a template for the next round of PCR reaction without DNA recovery.
The following is an example of 15 sets of primers designed in the first step, and the specific synthesis steps are as follows:
1. round 1 PCR
And respectively carrying out the 1 st round of overlay PCR amplification on the 15 groups of primers simultaneously to obtain 1 st round of PCR products, and obtaining 15 fragments in total. The fragment obtained from the 1 st primer set (F1, F-T1, R1 and R-T1) was designated as fragment 1, the fragment obtained from the 2 nd primer set (F2, F-T2, R2 and R-T2) was designated as fragment 2, … …, and so forth, and the fragment obtained from the 15 th primer set (F15, F-T15, R15 and R-T15) was designated as fragment 15.
Using the 1 st primer set as an example, an overlay PCR amplification reaction system (15. Mu.l) was prepared according to Table 2, and after the reaction system was prepared, the reaction was performed in a PCR apparatus at a high fidelity enzyme extension rate of 1kb/min, and the reaction procedure was as shown in Table 3.
TABLE 2
| Component (A) | Volume (mul) |
| Stencil (F-T1, R-T1) | 0.5 μl each (10 μM) |
| Primer (F1, R1) | 0.5 μl each (10 μM) |
| Phanta Max Super-Fidelity DNA Polymerase | 0.25μl |
| 2x Phanta Max Buffer | 7.5μl |
| dNTP mix | 0.5μl |
| PCR enhancer | 3μl |
| ddH 2 O | 1.75μl |
TABLE 3 Table 3
2. Round 2 PCR
And (3) carrying out 2 nd round of overlay PCR amplification by taking the 1 st round of PCR products of the adjacent 2 groups obtained in the step (1) as templates to obtain 7 fragments in total.
Wherein, take fragment 1 and fragment 2 as template to carry on 2 nd round of overlay PCR amplification, get fragment 1+2. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 3 and 4 as templates to obtain the fragments 3+4. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 5 and 6 as templates to obtain the fragments 5+6. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 7 and 8 as templates to obtain the fragments 7+8. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 9 and 10 as templates to obtain the fragments 9+10. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 11 and 12 as templates to obtain the fragments 11+12. And carrying out 2 nd round of overlay PCR amplification by taking the fragments 13 and 14 as templates to obtain the fragments 13 and 14.
Using the example of the fragments 1 and 2, a PCR reaction system (15. Mu.l) was prepared according to Table 4, and after preparing the reaction system, the reaction was carried out in a PCR apparatus at a high-fidelity enzyme extension rate of 1kb/min, and the reaction procedure was as shown in Table 3.
TABLE 4 Table 4
| Component | Volume(μl) |
| Stencil (fragment 1, fragment 2) | 1 μl each |
| Primer (F1, R2) | 0.5 μl each (10 μM) |
| Phanta Max Super-Fidelity DNAPolymerase | 0.25μl |
| 2x Phanta Max Buffer | 7.5μl |
| dNTP mix | 0.5μl |
| PCR enhancer | 3μl |
| ddH 2 O | 0.75μl |
3. Round 3 PCR
And (3) performing 3 rd round of overlay PCR amplification by taking the 2 nd round of PCR products of the two adjacent groups obtained in the step (2) as templates to obtain the 3 rd round of PCR products. And 3 rd round of overlay PCR amplification is carried out by taking the fragments 1+2 and 3+4 as templates, so as to obtain the fragments 1-4. And 3 rd round of overlay PCR amplification is carried out by taking the fragments 5+6 and 7+8 as templates, so as to obtain the fragments 5-8. And 3 rd round of overlay PCR amplification is carried out by taking the fragments 9+10 and 11+12 as templates, so as to obtain the fragments 9-12. And 3 rd round of overlay PCR amplification is carried out by taking the fragments 13+14 and 15 as templates, so as to obtain the fragments 13-15.
Using the fragments 1+2 and 3+4 as examples, a PCR reaction system (15. Mu.l) was prepared according to Table 5, and after preparing the reaction system, the reaction was carried out in a PCR apparatus at a high fidelity enzyme extension rate of 1kb/min, and the reaction procedure was as shown in Table 6.
TABLE 5
| Component (A) | Volume (mul) |
| Stencil (fragment 1+2, fragment 3+4) | 1 μl each |
| Primer (F1, R4) | 0.5 μl each (10 μM) |
| Phanta Max Super-Fidelity DNAPolymerase | 0.25μl |
| 2x Phanta Max Buffer | 7.5μl |
| dNTP mix | 0.5μl |
| PCR enhancer | 3μl |
| ddH 2 O | 0.75μl |
TABLE 6
4. Round 4 PCR
And (3) carrying out 4-round overlay PCR amplification by taking the 3 rd round PCR products of the two adjacent groups obtained in the step (3) as templates to obtain 4-round PCR amplification. And 4. The 4 th round of overlay PCR amplification is carried out by taking the fragments 1-4 and the fragments 5-8 as templates, so as to obtain the fragments 1-8. And 4 th round of overlay PCR amplification is carried out by taking the fragments 9-12 and the fragments 13-15 as templates, so as to obtain the fragments 9-15.
Taking fragments 1-4 and fragments 5-8 as examples, a PCR reaction system (15. Mu.l) was prepared according to Table 7, and after preparing the reaction system, the reaction was carried out in a PCR apparatus at a high fidelity enzyme extension rate of 1kb/min, and the reaction procedure was as shown in Table 6.
TABLE 7
| Component (A) | Volume (mul) |
| Stencil (fragment 1-4, fragment 5-8) | 1 μl each |
| Primer (F1, R8) | 0.5 μl each (10 μM) |
| Phanta Max Super-Fidelity DNAPolymerase | 0.25μl |
| 2x Phanta Max Buffer | 7.5μl |
| dNTP mix | 0.5μl |
| PCR enhancer | 3μl |
| ddH 2 O | 0.75μl |
5. Round 5 PCR
And (3) carrying out 5 th round of overlay PCR amplification by taking the 4 th round of PCR products of the two adjacent groups obtained in the step (4) as templates to obtain 5 th round of PCR products.
And carrying out 5 th round of overlay PCR amplification by taking the fragments 1-8 and the fragments 9-15 as templates to obtain the full-length gene fragments 1-15. The final round of PCR was thus 1 round, and recovery and purification of the PCR product were required, so that the PCR system was expanded to 50. Mu.l/tube, 3 tubes, and 150. Mu.l total. A PCR reaction system (50. Mu.l) was prepared in accordance with Table 8, and after the preparation of the reaction system, the reaction was carried out in a PCR apparatus at a high-fidelity enzyme extension rate of 1kb/min, and the reaction procedure was as shown in Table 9.
TABLE 8
| Component (A) | Volume (mul) |
| Stencil (fragment 1-8, fragment 9-15) | 3 μl each |
| Primer (F1, R15) | 2 μl each (10 μM) |
| Phanta Max Super-Fidelity DNA Polymerase | 0.5μl |
| 2x Phanta Max Buffer | 25μl |
| dNTP mix | 1μl |
| PCR enhancer | 10μl |
| ddH 2 O | 3.5μl |
TABLE 9
3. Cloning of PCR products into vectors
1. And (3) recovering and purifying the PCR product finally obtained in the step two from agarose gel by using a silica gel column purification technology to obtain a purified PCR product.
2. Linearization is performed on the target gene expression vector pEG BacMAM (Addgene, product number 160451) by using Template-F and Template-R to obtain a linearization vector.
Template-F: taatttcgaatctagagcctgcag (24 bp base complementary pairing to the target gene primer R15);
Template-R: ggacatcgtccacggtggtgggcatgaattcgggcccctggaaca (24 bp base complementary pairing to the target gene primer F1).
3. And (3) connecting the linearization vector obtained in the step (2) with the purified PCR product obtained in the step (1) through a homology arm to obtain the recombinant plasmid. The carrier dosage is as follows: 0.02 number of carrier bases per gum recovery product concentration, amount of fragment of interest: 0.04 number of gene bases/concentration of gel recovered product, adding recombinase (2 x ClonExpress Mix), and then placing in a PCR instrument for reaction, wherein the reaction procedure is 50 ℃, 15min,4 ℃ and infinity, and the reaction is completed in the PCR instrument.
4. And (3) transforming the recombinant plasmid obtained in the step (3) into escherichia coli DH5 alpha competent cells by using a heat shock transformation method. A proper amount of bacterial liquid is uniformly coated on an ampicillin resistant plate, and the plate is placed in an incubator at 37 ℃ for overnight culture.
4. Sequencing verification
Utilizing LB plate culture coated with ampicillin, selecting a monoclonal colony after overnight culture and performing colony PCR, and performing preliminary sequence verification (sequencing verification on the amplified sequence); cloning colony PCR positive, amplifying culture, extracting plasmid, purifying, and gene sequencing to verify, so as to ensure no mutation, SNPs, insertion or deletion of gene and complete gene synthesis.
The verification result shows that: the invention successfully synthesizes a long fragment gene SLC22A2 with the length of 1665bp shown in the sequence 1, and the whole process of the gene synthesis method takes 2-3 days.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Claims (10)
1. A method for synthesizing a long fragment gene comprises the following steps:
1) Dividing a target long fragment gene sequence into m sections, and then designing an overlay PCR primer for each section to obtain m groups of primers; each group of primers comprises 2 forward primers and 2 reverse primers, the 2 forward primers are respectively marked as a primer F and a primer F-T, the 2 reverse primers are respectively marked as a primer R and a primer R-T, wherein the primer F-T is a reverse complementary sequence of the primer R-T, the two primers are both used as templates for the overlay PCR amplification, an overlapping section is arranged between the 2 forward primers, an overlapping section is arranged between the 2 reverse primers, and an overlapping section is arranged between two adjacent groups of primers;
2) Respectively carrying out the 1 st round of over lap PCR amplification on each group of primers at the same time to obtain m over lap PCR amplification products; then, mixing two adjacent sets of the overlay PCR amplification products, and simultaneously carrying out 2 nd round of overlay PCR amplification to synthesize small fragments in the two adjacent sets of the overlay PCR amplification products into large fragments; and by analogy, obtaining a final overlay PCR amplification product by multiple rounds of overlay PCR amplification, namely the long fragment gene.
2. The method according to claim 1, characterized in that: in the step 1), the length of each primer in each group of primers is less than or equal to 59bp.
3. The method according to claim 1 or 2, characterized in that: in the step 1), the effective extension fragment length of each primer set is 110-120bp.
4. A method according to any one of claims 1-3, characterized in that: in the step 1), the length of the overlapped segment is 20-25bp.
5. The method according to any one of claims 1-4, wherein: in the step 2), the over lap PCR amplification product of the previous round is directly used as a template for the reaction of the over lap PCR amplification product of the next round without DNA recovery and purification.
6. The method according to any one of claims 1-5, wherein: in the step 2), the final Overlap PCR amplification product has a length of (2) n-1 X 115) bp, wherein n is the number of amplification rounds.
7. The method according to any one of claims 1-6, wherein: the method further comprises the steps of cloning the final overlay PCR amplification product into an expression vector and sequencing and verifying the obtained recombinant vector.
8. The method according to claim 7, wherein: the method for cloning the final overlay PCR amplification product to an expression vector and sequencing and verifying the obtained recombinant vector comprises the following steps:
a. recovering and purifying the final overlay PCR amplification product to obtain a purified final overlay PCR amplification product;
b. cloning the purified final Overlap PCR amplification product into an expression vector to obtain a recombinant vector;
c. transforming the recombinant vector into escherichia coli, culturing overnight, selecting a monoclonal colony, and performing colony PCR verification to obtain colony PCR positive clones;
d. and (3) amplifying and culturing the colony PCR positive clone, extracting and purifying plasmids, and verifying by adopting a gene sequencing technology to complete gene synthesis.
9. The method according to any one of claims 1-8, wherein: the target long fragment gene sequence is shown as a sequence 1.
10. Use of the method of any one of claims 1-9 for increasing the efficiency of long fragment gene synthesis or for shortening the long fragment gene synthesis time and/or for reducing the cost of long fragment gene synthesis.
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