CN107760742B - Synthesis method of gene rich in AT or GC - Google Patents
Synthesis method of gene rich in AT or GC Download PDFInfo
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Abstract
The invention discloses a synthetic method of genes rich in AT or GC. The method comprises the following steps: 1) Dividing two strands of a gene into a plurality of oligonucleotide fragments respectively, wherein one strand oligonucleotide fragment and the corresponding other strand oligonucleotide fragment can be complementarily paired to generate a double-stranded oligonucleotide fragment with a sticky end; the adjacent cohesive ends of two adjacent double-stranded oligonucleotide fragments can be complementarily paired, and the lengths of the cohesive ends are 4,6 and … (n + 1) bases in sequence according to the sequence of the gene; 2) Synthesizing oligonucleotide fragments; 3) Slowly annealing the oligonucleotide chain in sections to form double-stranded oligonucleotide fragments; 4) Phosphorylation of the double-stranded oligonucleotide fragment; 5) The double-stranded oligonucleotide fragments are ligated into a complete gene. The method achieves efficient assembly of AT-or GC-rich genes.
Description
Technical Field
The invention relates to the field of bioengineering, in particular to a synthesis method of genes rich in AT or GC.
Technical Field
With the development of medical engineering, bioengineering and synthetic biology, there is an increasing demand for specifically performing in vitro DNA synthesis (gene synthesis) based on naturally occurring DNA sequences or artificially created DNA sequences. In vitro gene synthesis can realize codon combination of gene optimization according to the codon characteristics of protein, thereby realizing high-efficiency expression of the protein. It can also realize multigene fusion and effective fusion of genes and multiple regulatory regions according to the research and development requirements. Recently, in vitro gene synthesis has also been used to create novel nanomaterials based on DNA.
Currently, a DNA synthesis method based on a Polymerase Chain Reaction (PCR) technique is widely used in the industry. This synthesis is performed by first dividing a gene into several short single-stranded oligonucleotide fragments, either manually or using software. Wherein a portion of the single-stranded oligonucleotide fragments are located on the sense strand of the gene and a portion of the single-stranded oligonucleotide fragments are located on the antisense strand of the gene; the single-stranded oligonucleotide fragments on the sense and antisense strands are bridged across the entire length of the gene by a length of complementary paired overlap. These single-stranded oligonucleotide fragments are then synthesized using a chemical synthesis route including a solid phase synthesis method, a phosphodiester method, a phosphotriester method, a phosphite triester method, a chip synthesis method, etc., using the modified base as a starting material (it should be noted that the longer the length of the single-stranded DNA oligonucleotide fragment is, the higher the error rate is, the better the fidelity is at a length of 100bp or less in the single-stranded DNA oligonucleotide synthesis techniques in the industry at present, and therefore, at present, the length is usually controlled to be within 100bp at the stage of chemical synthesis in various specific gene synthesis methods). Then, oligonucleotide fragments of the bridged sense strand and antisense strand are used as templates, and PCR technology is used for one-round or multiple-round amplification to obtain a large amount of complete gene DNA. These DNAs are finally loaded onto plasmid vectors by digestion, enzymatic ligation or other molecular cloning means, transformed into E.coli, verified by sequencing and further amplified in large quantities. Some specific methods, although differing in PCR procedures or some primer designs, essentially cover the core procedures described above (references include Stemmer et al, 1995, kodumalet al, 2004 reisinger et al, 2006, xiong et al, 2004 and Xiong et al, 2006, etc..
The gene synthesis method based on the PCR technology can effectively synthesize genes with GC content of 40-60%, but has limited synthesis capability for some genes rich in AT (low GC) or high GC. The lower the GC or about the higher the GC, the more difficult the synthesis. For AT contents above 80% or GC contents above 70%, PCR techniques are hardly able to achieve efficient synthesis. Generally, synthesis by PCR is GC-rich, often resulting in synthesis failure because of the tendency of GC hairpins to form between oligonucleotide strands or local DNA products. In contrast, when an AT-rich gene is synthesized by PCR, synthesis failure often results from difficulty in forming a stable DNA double strand. In addition, GC-rich and AT-rich genes are often accompanied by high repeats that result in synthetic gene deletions or insertions that result in synthetic failure.
Another gene synthesis technique that has been used in small quantities is a gene assembly synthesis technique based on a ligase method. This method is actually a more ancient gene synthesis technique than the PCR method. In contrast to the PCR method described above, it also requires that short single-stranded oligonucleotide fragments be first designed and chemically synthesized, with only slight differences in the design of the specific oligonucleotide strands. Specifically, the method can be roughly divided into the following steps: 1) First, a gene is divided into several short single-stranded oligonucleotide fragments, either manually or using software. Wherein half of the single-stranded oligonucleotide fragment is located on the sense strand of the gene and half of the single-stranded oligonucleotide fragment is located on the antisense strand of the gene; certain staggered and overlapped complementary regions are kept between the oligonucleotide segments positioned on the sense strand and the oligonucleotide segments positioned on the antisense strand; the length of the overlapping complementary region is random; finally, the single-stranded oligonucleotide fragments on the sense strand and the antisense strand can form a complete double-stranded DNA only lacking phosphodiester linkage between oligonucleotides through complementary pairing. 2) The single-stranded oligonucleotide fragment was synthesized by the same method as the method for synthesizing a single-stranded oligonucleotide as in the PCR synthesis method described above. 3) The obtained single-stranded oligonucleotide fragment was subjected to phosphorylation treatment. 4) Mixing multiple single-stranded oligonucleotide fragments of sense strand and antisense strand of the obtained gene in the same system, annealing, adding ligase, and connecting to form a complete double strand. The resulting DNA duplex can be further stored and amplified in large quantities by molecular cloning means on plasmid vectors (references include Gupta et al, 1968, hsiung et al, 1979, eren and Swenson,1989, grund strom et al, 1985 and Au et al, 1998, etc.). The method can also be combined with PCR to improve synthesis ability. The complete double-stranded DNA obtained by step 4 can be amplified using PCR techniques to increase the yield of synthesis and the length of gene synthesis (references include Smith et al, 1990.
Based on the DNA synthesis technique by the conventional ligase method, it is also possible to synthesize a gene having a GC content of 40% to 60%. However, for low GC and high GC genes, the synthesis capacity is even lower than if PCR technology alone was relied upon for synthesis. Among the reasons include 1) the heterogeneity of local GC contents of high GC and low GC genes, which prevents the formation of a complete DNA double strand, because a plurality of single-stranded oligonucleotides mixed in the same system form local secondary structures themselves during annealing. 2) Due to the high repeat, sense strand, and oligonucleotide pairs on the antisense strand that are themselves mismatched with each other by the high GC and low GC genes, synthesis failure cannot be ruled out by the subsequent screening process. 3) Due to the high repetition associated with the high GC and low GC genes, cohesive ends of the sense strand, which are generated by DNA duplexes formed by antisense strand oligonucleotides, which are partially identical in length, can be mismatched with each other, resulting in synthesis failure.
In view of the low synthesis ability of the existing methods for the GC-and AT-rich genes, the GC-or AT-rich genes are defined as difficult genes by the respective large gene synthesis companies, and are distinguished from the standard services thereof (see websites of the respective large gene synthesis companies). Although there are some differences in the definition criteria for GC-or AT-rich difficulty genes, the more classical approach is to define genes with a GC-or AT-content higher than 60% as difficulty genes. About 30% of these difficult genes, each gene synthesis company, may take 3 to 10 times as long as the normal genes to synthesize by repeatedly optimizing the processes of the PCR method and the ligase method. The remaining approximately 70% of the genes, each large gene company, will fail to provide service due to repeated attempts.
Disclosure of Invention
The invention aims to solve the problem that the synthesis of the existing gene rich in AT or GC is difficult, and provides an effective synthesis method of the gene rich in AT or GC.
The invention is summarized as follows (as shown in the flow chart of the attached figure 1):
a synthetic method of a synthetic method of AT or GC rich genes comprises the following steps:
the method comprises the following steps: dividing one chain of the gene into a plurality of oligonucleotide fragments of A1, A2 … At; dividing the other strand of the gene into a plurality of oligonucleotide fragments of B1, B2 … Bt; the designed An and the corresponding Bn oligonucleotide fragment can be complementarily paired to generate a double-stranded oligonucleotide fragment with sticky ends, wherein the adjacent sticky ends of two adjacent double-stranded oligonucleotide fragments can be complementarily paired, and the length of the sticky ends is 4,6,8 and 10 … (n + 1) bases in sequence according to the sequence order of the gene; n is selected from 1,2,3, … …, t;
step two, obtaining the oligonucleotide fragment designed in the step one;
step three: respectively carrying out segmentation slow annealing on the oligonucleotide chains of An and Bn to form double-stranded oligonucleotide fragments;
step four: phosphorylating the double-stranded oligonucleotide fragment obtained in step 3 with phosphorylase;
step five: and (4) connecting the double-stranded oligonucleotide fragments obtained in the fourth step into a complete gene by using ligase.
The AT-rich or GC-rich gene refers to a gene with an AT content of more than 60% or a gene with a GC content of more than 60% in the gene.
T is preferably an integer of 1 or more and 15 or less.
The first step preferably comprises: dividing one chain of gene into a plurality of oligonucleotide fragments of A1, A2 … At manually or by using software; dividing the other chain of the gene into a plurality of oligonucleotide fragments of B1, B2 … Bt manually or by using software; an and the corresponding Bn oligonucleotide fragment are designed to be capable of complementary pairing to generate a double-stranded oligonucleotide fragment with a sticky end, wherein the sticky end at the 3 'end of the double-stranded oligonucleotide fragment An/Bn and the sticky end at the 5' end of the double-stranded oligonucleotide fragment An +1/Bn +1 can be complementarily paired in the direction of An, and the sticky end is 2 (n + 1) bases in length.
The length of the oligonucleotide fragment in step one is preferably 20-150bp.
The method for obtaining the oligonucleotide fragment designed in the step one in the step two is any one of the existing methods for synthesizing oligonucleotide fragments, preferably a chemical synthesis method; the chemical synthesis methods involved include, but are not limited to, solid phase synthesis methods, phosphodiester methods, phosphotriester methods, and chip synthesis methods.
The step three of the stepwise slow annealing preferably separately anneals the two oligonucleotide fragments of each double-stranded oligonucleotide fragment synthesized in a centrifuge tube, i.e., different oligonucleotide pairs An/Bn are added to different reaction tubes and annealed separately. Comprises firstly, denaturing oligonucleotide pairs at 85-95 deg.C for 5-15min, and then naturally cooling the denatured oligonucleotide pairs at a temperature lower than 30 deg.C, or slowly cooling and annealing at a temperature of 0.1-2 deg.C per minute.
In the method of the present invention, the annealing of the oligonucleotide fragment further preferably comprises the steps of: 1ul oligonucleotide fragment An,1ul oligonucleotide fragment Bn,10 ul2 XSSC buffer, 8ul H 2 Adding O into a 1.5ml reaction tube, uniformly mixing, performing denaturation at 95 ℃ for 5min, and cooling at room temperature for 30min-1h; wherein the initial concentration of the oligonucleotide fragment An and the oligonucleotide fragment Bn are both 50uM.
The phosphorylation in step four preferably comprises the steps of: taking 1ul of each double-stranded oligonucleotide fragment A1/B1, A2/B2 … An/Bn formed by annealing in the third step, adding T4PNK kinase 2ul and T4PNK kinase buffer 2ul, supplementing water to 20ul, and reacting at 37 ℃ for 0.5h.
In the method, the 5 'end of the double-stranded oligonucleotide fragment A1/B1 and the 3' end of At/Bt can be respectively designed to generate a flat end or a sticky end with a target vector for cloning a specific restriction endonuclease on the vector according to subsequent cloning requirements.
Step five involves ligating the phosphorylated double-stranded oligonucleotide fragments into a complete gene using any ligase, such as T4 ligase.
The ligation process of the phosphorylated product preferably comprises the following steps: from the annealed phosphorylated product5ul of the vector was added to 1ul of the digested target vector, 10ul of 2 Xligase buffer,3ul of H 2 O, reacting at room temperature for 0.5h.
The method comprises the following steps that when the 5 'end of the double-stranded oligonucleotide fragment A1/B1 and the 3' end of At/Bt are designed to generate flat ends, the connection process of the phosphorylation products after annealing comprises the following steps: 5ul of the annealed phosphorylated product was added to 1ul of the linearized blunt-ended target vector, 10ul of 2 Xligase buffer,3ul of H2O, and the reaction was carried out at room temperature for 0.5h.
The linearized blunt-ended target vector may be an EcoRV cleavage or PCR amplification.
Has the advantages that:
the method has the advantages that: the method provided by the invention can effectively synthesize AT (content is equal to 60%) or GC (content is equal to 60%), and makes up for the technical shortage that the AT or GC rich gene cannot be synthesized or is difficult to synthesize by using the traditional method in the industry. The method is an efficient method that enables synthesis of a large number of single-stranded oligonucleotide-length AT and GC-rich genes in one synthesis cycle (encompassing the procedures of gene DNA synthesis, transformation into E.coli for amplification, sequencing verification, etc., usually 2-6 days), and in some cases, even more efficiently than normal genes (40% -60% GC content) as currently defined in the industry. The method of the invention, complementary single-stranded oligonucleotides, separately slow annealing, ensures that all single-stranded oligonucleotide segments covering genes rich in AT or GC can correctly form oligonucleotide double strands, and the design is the first key point for ensuring the correct synthesis of the genes rich in AT or GC. In the method of the present invention, the cohesive end pairing region of the double-stranded DNA formed by the single-stranded oligonucleotide is lengthened in the manner of 4,6,8, … (n + 1) in sequence. The selection of differential sticky end-linkers of even number of bases between such double-stranded oligonucleotide pairs enables the AT-or GC-rich double-stranded oligonucleotide pairs to synthesize genes correctly according to the sequence order of the genes. In addition, the unique design makes it impossible to join the cohesive ends of a double-stranded oligonucleotide pair into a correct gene in the case of a mismatch due to GC-rich or AT-rich repeats. For example, a mismatch of sticky ends of 4 bases and 6 bases leaves a2 base gap. Through experimental optimization, the design of the obtained differential sticky end joint with even number of bases is the most critical place for ensuring the synthesis of genes rich in AT or GC. The single-stranded oligonucleotide annealing program adopted by the invention can realize the annealing of the single-stranded oligonucleotide very simply and conveniently on the basis of not depending on expensive instruments. The method effectively avoids the complex procedures of glue running and recovery and a short plate for gene synthesis by PCR in the most classical gene synthesis PCR technology at present. Meanwhile, the technology avoids the problems that single-stranded oligonucleotides are easy to mismatch and cannot be annealed to form a complete gene when AT or GC rich genes are synthesized in the traditional ligase method, and realizes the effective assembly of the AT or GC rich genes. In conclusion, the AT or GC rich gene synthesis method provided by the invention can effectively synthesize AT or GC rich genes which cannot be synthesized or are difficult to synthesize by the traditional method, and has the advantages of unique design, simple and convenient operation and high synthesis efficiency.
Description of the drawings:
FIG. 1: the invention is a synthetic flow chart of AT or GC gene.
FIG. 2: sequencing results of example 1.
FIG. 3: sequencing results of example 2.
The specific implementation mode is as follows:
for a further understanding of the methods described herein, reference is made to the following description taken in conjunction with the accompanying drawings and examples.
Example 1:
the sequence of interest synthesized in this example was a sequence with an AT content of 93.7%. The sequence is shown as SEQ ID NO.1 in the sequence table.
The gene synthesis procedure of this example is as follows:
1) Manually dividing a sense strand of a gene into a plurality of oligonucleotide sequences of 70-80bp, marked as A1, A2, A3 and A4, and the sequences are sequentially shown as SEQ ID NO.2-5 in a sequence table; the antisense strand of the gene is manually divided into a plurality of oligonucleotide sequences of 70-80bp, marked as B1, B2, B3 and B4, and the sequences are shown as SEQ ID NO.6-9 in the sequence table in sequence.
Wherein A1 and B1 can be complementarily paired to form a cohesive end with CATT 4 bases at the 5' end of an antisense strand; a2 B2 can be complementarily paired to form a sticky end with 4 bases of AATG at the 5 'end of a sense strand, and a sticky end with 6 bases of TTTCTA at the 5' end of an antisense strand; a3 and B3 can be complementarily paired to form a sense strand with a sticky end with TAGAAA 6 bases at the 5 'end and a sticky end with TTTTTTCT at the 5' end; a4 B4 is capable of complementary pairing to form a sense strand with a cohesive end of AGAAAAAA at the 5' end.
2) Synthesizing the oligonucleotides A1, A2, A3 and A4 in the step 1 by using a chemical synthesis method; b1, B2, B3, B4, the concentration is 50uM.
3) 1ul A1,1ul B1 fragment, 10ul 2 XSSC buffer, 8ulH 2 Adding O into a 1.5ml reaction tube 1, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. The 1ul A2,1ul B2 fragment, 10ul 2 XSSC buffer, 8ulH were collected 2 Adding O into a 1.5ml reaction tube 2, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. 1ul A3,1ul B3 fragment, 10ul 2 XSSC buffer, 8ulH 2 Adding O into a 1.5ml reaction tube 3, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. The 1ul A4,1ul B4 fragment, 10ul 2 XSSC buffer, 8ulH were collected 2 Adding O into a 1.5ml reaction tube 4, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h.
4) 1ul of each reaction product was taken from the reaction tube 1,2,3,4 in step 3, added with T4PNK kinase 2ul and T4PNK kinase buffer 2ul, supplemented with water to 20ul, and reacted at 37 ℃ for 0.5hour.
5) 5ul of the phosphorylated product obtained in step 4 was added to 1ul of EcoRV-digested pUC57 vector, 10ul of 2 Xligase buffer, and 3ul of H 2 O reaction 0.5hour. 10ul of the ligation products were used to transform E.coli competent cells, top10, and LB plates with X-gal and IPTG were plated.
6) And (3) sequence synthesis detection: the positive clones obtained in 5 were screened for 3 and the extracted plasmids were sequenced and the sequence was completely correct (FIG. 2).
This example successfully achieved 93.7% AT gene synthesis in one synthesis cycle using the methods provided by the present invention. In this example, we also tried the conventional PCR method and ligase method repeatedly, and no correct sequence clone was obtained. This indicates that our method is of great advantage in AT-rich gene synthesis.
Example 2:
the sequence of interest synthesized in this example was a 72% sequence by GC. The sequence is shown as SEQ ID NO.10 in the sequence table.
The gene synthesis procedure of this example is as follows:
1) Manually dividing a sense strand of a gene into a plurality of oligonucleotide sequences of 50-60bp, wherein the oligonucleotide sequences are marked as A1, A2, A3 and A4, and the sequences are sequentially shown as SEQ ID NO.11-14 in a sequence table; the gene antisense strand is manually divided into a plurality of oligonucleotide sequences of 50-60bp, marked as B1, B2, B3 and B4, and the sequences are sequentially shown as SEQ ID NO.15-18 in the sequence table.
Wherein A1 and B1 can be complementarily paired to form a cohesive end with AGCTT 5 bases at the 5' end of a sense strand; the 5' end of the antisense strand is provided with a cohesive end with 4 bases GCCT; a2 B2 can be complementarily paired to form a sticky end with AGGC 4 bases at the 5 'end of a sense strand, and a sticky end with CCTGCC 6 bases at the 5' end of an antisense strand; a3 and B3 can be complementarily paired to form a sticky end with GGCAGG 6 bases at the 5 'end of a sense strand, and a sticky end with GACTACTC at the 5' end of an antisense strand; a4 B4 is capable of complementary pairing to form a sense strand with a cohesive end of GAGTAGTC at the 5 'end and an antisense strand with a cohesive end of AATTC at the 5' end. Wherein the 5 'end of the sense strand formed by complementary pairing of A1 and B1 has a cohesive end of AGCTT, and the 5' end of the antisense strand formed by complementary pairing of A4 and B4 has a cohesive end of AATTC, which can ensure that the fragment can be cloned to a vector cut by HindIII and EcoRI.
2) Synthesizing oligonucleotides A1, A2, A3 and A4 in the step 1 by using a chemical synthesis method; b1, B2, B3, B4, the concentration is 50uM.
3) 1ul A1,1ul B1 fragment, 10ul 2 XSSC buffer, 8ul H 2 Adding O into a 1.5ml reaction tube 1, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. 1ul A2,1ul B2 fragment, 10ul 2 XSSC buffer, 8ul H 2 Adding O into a 1.5ml reaction tube 2, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. 1ul A3,1ul B3 fragment, 10ul 2 XSSC buffer, 8ulH 2 Adding O into a 1.5ml reaction tube 3, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h. 1ul A4,1ul B4 fragment, 10ul 2 XSSC buffer, 8ulH 2 Adding O into a 1.5ml reaction tube 4, uniformly mixing, denaturing at 95 ℃ for 5min, and cooling at room temperature for 1h.
4) 1ul of reaction product is respectively taken from the reaction tube 1,2,3,4 in the step 3, T4PNK kinase 2ul and T4PNK kinase buffer 2ul are added, water is supplemented to 20ul, and the reaction is carried out for 0.5h at 37 ℃.
5) 5ul of the phosphorylated product obtained in step 4 was added to 1ul of HindIII-EcoRI-digested pUC57 vector, 10ul of 2 Xligase buffer, and 3ul of H 2 O reacts for 0.5h. 10ul of the ligation products were transformed into E.coli competent cells, top10, and plated on LB plates with X-gal and IPTG
6) And (3) sequence synthesis detection: screening 2 positive clones obtained in the step 5, extracting plasmid for sequencing, and completely correcting a sequencing result. The sequencing results are shown in FIG. 3.
This example successfully achieved the synthesis of genes containing 72% GC by one synthesis cycle using the method provided by the present invention. The gene, which we attempted to synthesize by conventional ligase method, all ended up with the wrong sequence. We also iteratively optimized the PCR program to synthesize, but after 3 cycles of synthesis failure, the correct sequence was still not obtained. This shows that our method has high success rate in the synthesis of GC-rich gene and great superiority.
Claims (9)
1. A method for synthesizing an AT-or GC-rich gene, comprising the steps of:
the method comprises the following steps: dividing a sense strand of a gene into a plurality of oligonucleotide fragments of A1, A2 … At manually or by using software; dividing the antisense strand of the gene into a plurality of oligonucleotide fragments of B1, B2 … Bt manually or by using software; the designed An and the corresponding Bn oligonucleotide fragment can be complementarily paired to generate a double-stranded oligonucleotide fragment with a sticky end, wherein the 3 'sticky end of the double-stranded oligonucleotide fragment An/Bn and the 5' sticky end of the double-stranded oligonucleotide fragment An +1/Bn +1 can be complementarily paired in the direction of the An, the sticky end is 2 (n + 1) bases in length, and n represents 1,2,3, … …, t;
step two, obtaining the oligonucleotide fragment in the step one;
step three: respectively carrying out segmentation slow annealing on the oligonucleotide chains of An and Bn to form double-stranded oligonucleotide fragments;
step four: phosphorylating the double-stranded oligonucleotide fragment obtained in the third step by using phosphorylase;
step five: connecting all the phosphorylated double-stranded oligonucleotide fragments obtained in the fourth step into a complete gene by using ligase;
wherein, the AT-rich or GC-rich gene refers to a gene with an AT content of more than 60% or a gene with a GC content of more than 60% in the gene.
2. The method according to claim 1, wherein t is an integer of 1 to 15 inclusive.
3. A method of synthesizing an AT or GC rich gene according to any of the claims 1~2 characterized in that the length of the oligonucleotide fragment is 20-150bp.
4. The method for synthesizing AT-or GC-rich gene according to claim 1 or 2, wherein the stepwise slow annealing in step three is performed by adding different oligonucleotide pairs An/Bn to different reaction tubes and annealing separately; comprises firstly, using high temperature of 85-95 ℃ to denature oligonucleotide pairs for 5-15min, then placing the denatured oligonucleotide pairs under the condition of lower than 30 ℃ for natural cooling, or slowly cooling and annealing in a mode of reducing 0.1-0.2 ℃ per minute.
5. The method for synthesizing AT-or GC-rich gene according to claim 4, wherein the annealing of the oligonucleotide fragment as described in claim 4 comprises the steps of: 1ul oligonucleotide fragment An,1ul oligonucleotide fragment Bn,10 ul2 XSSC buffer, 8ul H 2 Adding O into a 1.5ml reaction tube, uniformly mixing, performing denaturation at 95 ℃ for 5min, and cooling at room temperature for 30min-1h; wherein the initial concentration of oligonucleotide fragment An and oligonucleotide fragment Bn is 50uM.
6. The method of claim 2, wherein the phosphorylation step four comprises the following steps: taking 1ul from the double-stranded oligonucleotide fragments A1/B1 and A2/B2 … At/Bt formed by annealing in the third step, adding T4PNK kinase 2ul and T4PNK kinase buffer 2ul, supplementing water to 20ul, and reacting At 37 ℃ for 0.5h.
7. The method for synthesizing gene rich in AT or GC as claimed in claim 1, wherein the 5 'end of the double-stranded oligonucleotide fragment A1/B1 and the 3' end of AT/Bt are designed to generate blunt ends or sticky ends for cloning with specific restriction enzymes on the vector of interest respectively according to the subsequent cloning requirements.
8. The method for synthesizing gene rich in AT or GC according to claim 7, wherein the ligation of phosphorylation product comprises the following steps: 5ul is taken from the phosphorylation product, and added with a target vector digested by 1ul, 10ul 2 Xligase buffer,3ul H 2 O, reaction at room temperature 0.5h.
9. The method for synthesizing genes rich in AT or GC according to claim 7, wherein the ligation of phosphorylation products when the 5 'end of the double-stranded oligonucleotide fragment A1/B1 and the 3' end of AT/Bt are designed to generate blunt ends comprises the following steps: 5ul was taken from the phosphorylated product and added with 1ul linearized blunt end target vector, 10ul 2 Xligase buffer,3ul H 2 O, reaction at room temperature 0.5h.
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