CN113481194A - DNA synthesis method - Google Patents
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- CN113481194A CN113481194A CN202110553100.7A CN202110553100A CN113481194A CN 113481194 A CN113481194 A CN 113481194A CN 202110553100 A CN202110553100 A CN 202110553100A CN 113481194 A CN113481194 A CN 113481194A
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Abstract
The invention relates to the technical field of DNA synthesis, in particular to a DNA synthesis method. The DNA synthesis method of the present invention comprises: dividing a sequence of DNA to be synthesized into n sequence segments, wherein n is more than or equal to 2 and is an even number, the length of each sequence segment is 50-150bp, designing a primer aiming at each sequence segment, the primer comprises all sequences of the sequence segments or reverse complementary sequences of the sequences, the primer of the ith sequence segment is a forward primer, the primer of the (i + 1) th sequence segment is a reverse primer, a reverse complementary pairing region exists between the 3 'end of the primer of the ith sequence segment and the 3' end of the primer of the (i + 1) th sequence segment, and i is all odd numbers in the range of 1-n-1, and performing template-free PCR by using the primer pair to obtain a PCR product. The method can rapidly obtain a PCR product for vector construction and gene synthesis through one round of PCR, and has the advantages of simple and convenient operation, rapidness, high synthesis efficiency and high accuracy.
Description
Technical Field
The invention relates to the technical field of DNA synthesis, in particular to a DNA synthesis method.
Background
In recent years, PCR technology has been widely used in the field of vector construction and gene synthesis. In the existing gene synthesis PCR method of multiple primers, PCR after primer mixing needs more than 2 PCR cycles, and finally, PCR products are obtained for molecular cloning experiments. In the method, a plurality of primers are mixed to obtain a template sequence through a first round of PCR, and on the basis, a product obtained by the first round of PCR is used as a template to perform a second round of PCR so as to obtain a PCR product of a target synthetic sequence. When the sequence to be synthesized has high GC content or contains a small amount of repetitive sequences, the PCR efficiency of the method is obviously reduced, so that the product acquisition fails or the deletion is found by sequencing after cloning.
The currently employed methods for synthesizing genes by annealing have the following problems: (1) the result of the annealing experiment cannot be directly and accurately analyzed, and the effect of the annealing experiment cannot be detected by a simple and effective detection method (such as electrophoresis) after the annealing experiment is finished; (2) the purity of a target sequence product obtained by an annealing experiment is usually low, secondary structure products among primers exist in an annealing system and cannot be separated basically in an electrophoresis mode, so that the target sequence product is difficult to purify effectively after annealing, and the subsequent experiment efficiency is influenced; (3) the product obtained by the annealing method can only be used for enzyme digestion and connection experiments basically, is difficult to be applied to homologous recombination and has limitation. Compared with annealing experiments, products of PCR experiments can be effectively applied to enzyme digestion connection and homologous recombination. Therefore, it is important to develop a simple and rapid method for gene synthesis using PCR technology.
Disclosure of Invention
The invention aims to provide a DNA synthesis method. The method can obtain PCR products by using one round of PCR for molecular cloning to synthesize DNA.
Specifically, the invention provides the following technical scheme:
the invention provides a DNA synthesis method, which comprises the following steps:
dividing a sequence of DNA to be synthesized into n sequence segments, wherein n is more than or equal to 2 and is an even number, the length of each sequence segment is 50-150bp, designing a primer aiming at each sequence segment, wherein the primer comprises all sequences of the sequence segments or reverse complementary sequences thereof,
in the primers, the primer of the ith sequence fragment is a forward primer, the primer of the (i + 1) th sequence fragment is a reverse primer, a first reverse complementary pairing region exists between the 3 'end of the primer of the ith sequence fragment and the 3' end of the primer of the (i + 1) th sequence fragment, wherein i is all odd numbers in the range of 1-n-1,
carrying out PCR amplification by using a primer pair consisting of the primer of the ith sequence segment and the primer of the (i + 1) th sequence segment to obtain a PCR product,
the reaction system for PCR amplification does not contain template DNA.
The invention divides the sequence of the DNA to be synthesized into short sequence segments, designs specific primers aiming at each short sequence segment, and utilizes the primer pairs of two adjacent short sequence segments to carry out PCR amplification, thereby obtaining a PCR product covering the complete sequence of the two adjacent short sequence segments.
Preferably, the length of the first reverse complementary region is 12-25bp, more preferably 16-22 bp. The length of the complementary pairing region of the primers of the two adjacent short sequence fragments is controlled in the range, so that the amplification efficiency of PCR can be obviously improved.
For the primers described above, there is a second reverse complementary paired region at the 5 'end of the primer of the x-th sequence segment and the 5' end of the primer of the x + 1-th sequence segment, wherein x is all even numbers in the range of 2 to n-2.
The second reverse complementary pairing region described above is used for homologous recombination of different PCR products.
Preferably, the length of the second reverse complementary pairing region is 12-80bp, more preferably 16-75 bp. Controlling the length of the second reverse complementary pairing region within the above range can significantly improve the efficiency of homologous recombination between PCR products.
Preferably, the Tm of the first reverse complementary pairing region is from 40 to 70 ℃.
The length of the complementary pairing region in the first direction can be adjusted within the range of 12-25bp in order to satisfy a Tm value of 40-70 ℃.
The number of complementary pairing bases of the first reverse complementary pairing region and other regions of the DNA to be synthesized except the first reverse complementary pairing region is less than or equal to 6. The invention discovers that the pairing of more than 6 bases can reduce the pairing efficiency between two primers, so that the effective amplification efficiency is reduced, and the probability of product deletion or amplification failure is increased.
Preferably, the PCR amplification comprises the steps of: pre-denaturation at 94-98 deg.C for 0.5-5min, denaturation at 94-98 deg.C for 10-20s, annealing at T deg.C for 10-20s, extension at 68-72 deg.C for 8-15s, and extension at 68-72 deg.C for 18-25 cycles, extension at 68-72 deg.C for 25-60s,
wherein the annealing temperature T is as follows:
when Tm is more than or equal to 65 ℃, T is Tm-5 ℃;
when Tm is more than or equal to 60 ℃ and less than 65 ℃, T is 60 ℃;
when Tm is more than or equal to 55 ℃ and less than 60 ℃, T is Tm;
when Tm < 55 ℃, T is 50 ℃;
tm is the Tm of the first reverse complementary paired region.
In the above PCR amplification procedure, the temperature for the pre-denaturation and extension can be adjusted within the above range according to the requirements of the DNA polymerase used.
The PCR amplification program is designed according to the characteristics of the primers, and the PCR amplification efficiency can be better ensured.
Preferably, the reaction system for PCR amplification comprises the following components: DNA polymerase, reaction buffer solution, primer pairs and dNTP, wherein the concentration of each primer in the primer pairs is 0.4-0.5 pmol/mu L in the reaction system. Controlling the primer concentration within this range can significantly improve the efficiency of PCR amplification using the primers of the present invention.
Further preferably, the reaction system for PCR amplification comprises the following components: 1 Xreaction buffer, 1-3U/. mu.L of DNA polymerase, 0.4-0.5 pmol/. mu.L of forward primer, 0.4-0.5 pmol/. mu.L of reverse primer, 0.1-0.3mM of dNTP, and water to make up the balance.
The DNA synthesis method also comprises a step of connecting the PCR products and a step of connecting the connection products of the PCR products with a carrier, wherein the connection between the PCR products is carried out by adopting a homologous recombination method.
The connection between the connection product of each PCR product and the vector can adopt an enzyme digestion connection method or a homologous recombination method. Preferably, the PCR products and the vector are ligated by one-step homologous recombination, wherein the 5' ends of the primer of the 1 st sequence segment and the primer of the n sequence segment also need to contain a region complementary to the sequence of the vector.
The DNA synthesis method provided by the invention can be suitable for synthesizing DNA with the length of 150-500bp, and is preferably more suitable for synthesizing DNA with the length of 150-380 bp.
The DNA synthesis method has no special requirements on the GC content of the DNA to be synthesized, and has higher synthesis efficiency on fragments with high GC content and repeated sequences.
The invention has the beneficial effects that: the DNA synthesis method provided by the invention realizes that a PCR product is quickly obtained for vector construction and gene synthesis through one round of PCR by utilizing complementary pairing between primers, and has the advantages of simple and convenient operation, quickness, high synthesis efficiency and high accuracy.
Compared with the synthesis method of multi-primer multi-round PCR amplification in the prior art, for the fragments with high GC content, the DNA synthesis method can realize high-efficiency PCR amplification without using high-specification PCR reagents aiming at the fragments with high GC content, and only needs one round of PCR amplification.
Compared with the method for synthesizing genes by annealing among a plurality of primers in the prior art, the DNA synthesis method has higher success rate and is more convenient to judge whether the DNA synthesis is successful or not.
Drawings
FIG. 1 shows the result of agarose gel electrophoresis detection of the PCR product of example 1 of the present invention, wherein lane 1 is DNA marker, lanes 4 and 5 are the products obtained by amplification with primers E1-F1 and E1-R1, and lanes 6 and 7 are the products obtained by amplification with primers E1-F2 and E1-R2.
FIG. 2 shows the results of the sequence verification of the product of example 1 of the present invention.
FIG. 3 shows the result of agarose gel electrophoresis detection of the PCR product of example 2 of the present invention, wherein Lane 1 is DNA marker, Lanes 2 and 3 are the products obtained by amplification with primers E2-F1 and E2-R1, and Lanes 4 and 5 are the products obtained by amplification with primers E2-F2 and E2-R2.
FIG. 4 shows the results of the sequence verification of the product in example 2 of the present invention.
FIG. 5 shows the result of electrophoretic detection of the PCR product in comparative example 1, wherein lane 1 is DNA marker, and lanes 2 and 3 are PCR amplification products.
FIG. 6 shows the results of the sequencing verification of the product of comparative example 2 according to the invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1
In the process of gene synthesis of large fragments, due to the existence of a specific structure of the sequence or other reasons, when the sequence of the synthesized fragment is analyzed, the obtained result is found to be inconsistent with the target sequence, and a sequence deletion of about 300bp is generated (SEQ ID NO. 1). Aiming at the sequence deletion of the small fragment, the DNA synthesis method provided by the invention is adopted to quickly obtain the PCR product of the short fragment, and the homologous recombination method is combined to obtain the plasmid connected with the deletion sequence of the fragment, and the specific method is as follows:
(1) dividing the sequence shown in SEQ ID NO.1 into 4 sequence segments, and sequentially marking as a sequence segment 1, a sequence segment 2, a sequence segment 3 and a sequence segment 4;
(2) designing a primer aiming at each sequence fragment, and designing 4 long-chain primers in total, wherein the primer sequence is as follows, wherein the lower case part is a vector homologous region, 18bp at the 5 'end of E1-R1 and E1-F2 is a second reverse complementary pairing region, and 17bp at the 3' end of E1-F1, E1-R1, E1-F2 and E1-R2 is a first reverse complementary pairing region.
Forward primer E1-F1 binds to sequence fragment 1 (5 '-3', SEQ ID NO. 2):
ccaagctggctagttaagcttggtaccATGGTGGCCGGGACCCGCTGTCTTCTAGCGTTGCTGCTTCCCCAGGTCCTCCTGGGCGGCGCGGCTGGCCTCGTTC；
the reverse primer E1-R1 binds to sequence fragment 2 (5 '-3', SEQ ID NO. 3):
CTCGCTCAGGACCTCGTCAGAGGGCTGGGATGAGGGGCGGCCCGACGACGCCGCCGCGAACTTCCTGCGGCCCAGCTCCGGAACGAGGCCAGCCGCG；
forward primer E1-F2 binds to sequence fragment 3 (5 '-3', SEQ ID NO. 4):
GACGAGGTCCTGAGCGAGTTCGAGTTGCGGCTGCTCAGCATGTTCGGCCTGAAACAGAGACCCACCCCCAGCAGGGACGCCGTGGTGCCCCCCTACATGCTAG；
the reverse primer E1-R2 binds to sequence fragment 4 (5 '-3', SEQ ID NO. 5):
tgttcgaagggccctctagactcgaggatatcgaattcGTCTGGGGCGGGTGAGCCCGGCTGACCTGAGTGCCTGCGATACAGGTCTAGCATGTAGGGGGGC。
in the above primers, E1-F1/E1-R1 is a group of primers, and the 3' ends of E1-F1 and E1-R1 have a first reverse complementary pairing region, and the parameters are as follows: length 17bp, GC content 76%, Tm 65.5 ℃; E1-F2/E1-R2 is a group of primers, and a first reverse complementary pairing region exists at the 3' end of E1-F2 and E1-R2, and the parameters are as follows: length 17bp, GC content 64%, Tm 53.1 ℃.
A second reverse complementary pairing region is present at the 5' end of E1-R1 and E1-F2, and the length of the second reverse complementary pairing region is 18 bp.
(3) PCR amplification
E1-F1, E1-R1, E1-F2 and E1-R2 are respectively diluted to 20-25 pmol/microliter for PCR amplification, and a PCR template is not required to be added into a PCR system.
The reaction system for PCR amplification of E1-F1/E1-R1 is as follows: 10 XPV 2 buffer 5. mu.l, E1-F1 primer 1. mu.l, E1-R1 primer 1. mu.l, polymerase PV 21. mu.l, dNTP (10mM) 1. mu.l, ddH2O 41μl。
The reaction procedure for PCR amplification was as follows:
the reaction system for PCR amplification of E1-F2/E1-R2 is as follows: 10 XPV 2 buffer 5. mu.l, E1-F2 primer 1. mu.l, E1-R2 primer 1. mu.l, polymerase PV 21. mu.l, dNTP (10mM) 1. mu.l, ddH2O 41μl。
The reaction procedure for PCR amplification was as follows:
the second round of PCR was not needed, and the results of electrophoresis detection of the two PCR products were shown in FIG. 1, after the PCR amplification was completed, agarose gel electrophoresis was performed.
(4) PCR product recovery
And (3) recovering two PCR products obtained by amplification of E1-F1/E1-R1 and E1-F2/E1-R2 according to the operation of the Axygen gel recovery kit specification, and using the recovered PCR products for downstream homologous recombination.
(5) Homologous recombination
The reaction system of homologous recombination is as follows: 3. mu.l of PCR product of E1-F1/E1-R1, 3. mu.l of PCR product of E1-F2/E1-R2, 10. mu.l of 2 XSoso clone kit, ddH2O 4μl。
Homologous recombination procedures were performed according to the Sosoo clone kit instructions.
(6) Molecular cloning
And (3) transforming host cells with the homologous recombination products, coating a flat plate, culturing overnight, selecting 8 single colonies for culturing the next day, extracting plasmids from all 8 single colonies, and randomly selecting 4 single colonies for sequencing verification.
Sequence deletion was confirmed by sequencing (fig. 2) in 2 of 4 plasmids sequenced, and the other 2 were in agreement with expectations and were identical to the target sequence.
Example 2
The target sequence to be synthesized is shown as SEQ ID NO.6, the PCR product of the short segment is quickly obtained by adopting the DNA synthesis method provided by the invention, and the plasmid connected with the segment sequence is obtained by combining a homologous recombination method, and the specific method is as follows:
(1) dividing the sequence shown in SEQ ID NO.6 into 4 sequence segments, and sequentially marking as a sequence segment 1, a sequence segment 2, a sequence segment 3 and a sequence segment 4;
(2) designing a primer aiming at each sequence fragment, and designing 4 long-chain primers in total, wherein the primer sequence is as follows, wherein a lower case part is a vector homologous region, the 5 ' end 48bp of E2-R1 and E2-F2 is a second reverse complementary pairing region, and the 3 ' end 21bp of E2-F1 and E2-R1 and the 3 ' end 17bp of E2-F2 and E2-R2 are first reverse complementary pairing regions.
Forward primer E2-F1 binds to sequence fragment 1 (5 '-3', SEQ ID NO. 7):
cgttgtaaaacgacggccagtgaattcCCCAATAAGCTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAAGGAAC；
the reverse primer E2-R1 binds to sequence fragment 2 (5 '-3', SEQ ID NO. 8):
CGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTTAAGATACATTGATG；
forward primer E2-F2 binds to sequence fragment 3 (5 '-3', SEQ ID NO. 9):
TCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAG；
the reverse primer E2-R2 binds to sequence fragment 4 (5 '-3', SEQ ID NO. 10):
gctatgaccatgattacgccgatatcaaacgacggccagtgaattaggTTAATTAAGGCTGCGCGCTCGCTCGCTCACTGAG。
in the above primers, E2-F1/E2-R1 is a group of primers, and the 3' ends of E2-F1 and E2-R1 have a first reverse complementary pairing region, and the parameters are as follows: length 21bp, GC content 33.3%, Tm 46.8 ℃; E2-F2/E2-R2 is a group of primers, and a first reverse complementary pairing region exists at the 3' end of E2-F2 and E2-R2, and the parameters are as follows: length 17bp, GC content 64.7%, Tm 51.9 ℃.
A second reverse complementary pairing region was present at the 5' end of E2-R1 and E2-F2, and the length of the second reverse complementary pairing region was 48 bp.
(3) PCR amplification
E2-F1, E2-R1, E2-F2 and E2-R2 are respectively diluted to 20-25 pmol/microliter for PCR amplification, and a PCR template is not required to be added into a PCR system.
The reaction system for PCR amplification of E2-F1/E2-R1 is as follows: 10 XPV 2 buffer 5. mu.l, E2-F1 primer 1. mu.l, E2-R1 primer 1. mu.l, polymerase PV 21. mu.l, dNTP (10mM) 1. mu.ll，ddH2O 41μl。
The reaction procedure for PCR amplification was as follows:
the reaction system for PCR amplification of E2-F2/E2-R2 is as follows: 10 XPV 2 buffer 5. mu.l, E2-F2 primer 1. mu.l, E2-R2 primer 1. mu.l, polymerase PV 21. mu.l, dNTP (10mM) 1. mu.l, ddH2O 41μl。
The reaction procedure for PCR amplification was as follows:
the second round of PCR was not needed, and the results of electrophoresis detection of the two PCR products were shown in FIG. 3, after the completion of the above-mentioned PCR amplification, agarose gel electrophoresis was performed.
(4) PCR product recovery
And (3) recovering two PCR products obtained by amplification of E2-F1/E2-R1 and E2-F2/E2-R2 according to the operation of the Axygen gel recovery kit specification, and using the recovered PCR products for downstream homologous recombination.
(5) Homologous recombination
The reaction system of homologous recombination is as follows: 3. mu.l of PCR product of E2-F1/E2-R1, 3. mu.l of PCR product of E2-F2/E2-R2, 10. mu.l of 2 XSoso clone kit, ddH2O 4μl。
Homologous recombination procedures were performed according to the Sosoo clone kit instructions.
(6) Molecular cloning
And (3) transforming host cells with the homologous recombination products, coating a flat plate, culturing overnight, selecting 8 single colonies for culturing the next day, extracting plasmids from all 8 single colonies, and randomly selecting 4 single colonies for sequencing verification.
As proved by sequencing (FIG. 4), sequence deletion exists in 1 of 4 sequenced plasmids, and the other 3 plasmids accord with the expectation and are completely consistent with a target sequence.
The two examples are only used as examples, and the results prove that the PCR product obtained by the DNA synthesis method provided by the invention can be used for molecular cloning experiments, and the method can realize the synthesis of longer DNA fragments by adjusting the length of the segmented sequence fragments and the corresponding primers and matching with the homologous recombination technology.
Comparative example 1
The synthesis of the DNA fragment shown as SEQ ID NO.1 in example 1 was carried out by a conventional multi-primer multi-round PCR method.
The primer design scheme of the multi-primer and multi-round PCR method is as follows: primers 1-4(SEQ ID NO.11-14) were designed and synthesized, respectively, for PCR amplification. The PCR amplification results show that the primers 1-4 failed to amplify, and the expected product could not be obtained (FIG. 5). According to analysis, the GC content of the sequence is 69% on average, the GC content in individual regions is more than or equal to 71%, and the high GC content causes that the multi-primer multi-round PCR method cannot be used for amplification and synthesis of the sequence.
Comparative example 2
The DNA fragment shown in SEQ ID No.6 of example 2 was synthesized by a conventional multi-primer multi-round PCR method, which had the following primer design: 4 primers (SEQ ID NO.15-18) were designed and synthesized, respectively, for PCR amplification. The results showed that all PCR products yielded deletions as confirmed by sequencing (figure 6).
According to analysis, the GC% of the local sequence of the sequence is more than or equal to 71%, and the high GC content causes that the conventional PCR method is easy to generate deletion when being used for amplification and synthesis of the sequence.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (10)
1. A method for synthesizing DNA, comprising:
dividing the sequence of the DNA to be synthesized into n sequence segments, wherein n is more than or equal to 2 and is an even number, the length of the sequence segments is 50-150bp,
designing a primer for each sequence fragment, the primer comprising the entire sequence of the sequence fragment or the reverse complement thereof,
in the primers, the primer of the ith sequence fragment is a forward primer, the primer of the (i + 1) th sequence fragment is a reverse primer, a first reverse complementary pairing region exists between the 3 'end of the primer of the ith sequence fragment and the 3' end of the primer of the (i + 1) th sequence fragment, wherein i is all odd numbers within the range of 1-n-1,
carrying out PCR amplification by using a primer pair consisting of the primer of the ith sequence segment and the primer of the (i + 1) th sequence segment to obtain a PCR product,
the reaction system for PCR amplification does not contain template DNA.
2. The method for synthesizing DNA according to claim 1, wherein the length of the first reverse complementary region is 12 to 25 bp.
3. The method for synthesizing DNA according to claim 1 or 2, wherein in the primer, a second reverse complementary pairing region exists between the 5 'end of the primer of the x-th sequence segment and the 5' end of the primer of the x + 1-th sequence segment, wherein x is an even number in the range of 2 to n-2;
preferably, the length of the second reverse complementary pairing region is 12-80 bp.
4. The method for synthesizing DNA according to claim 3, wherein the Tm value of the first reverse complementary pairing region is 40 to 70 ℃.
5. The method of synthesizing DNA according to claim 3 or 4, wherein the number of complementary pairing bases of the first reverse complementary pairing region and the region other than the first reverse complementary pairing region to be synthesized is 6 or less.
6. The method for synthesizing DNA according to any one of claims 1 to 5, wherein the PCR amplification comprises the steps of: pre-denaturation at 94-98 deg.C for 0.5-5min, denaturation at 94-98 deg.C for 10-20s, annealing at T deg.C for 10-20s, extension at 68-72 deg.C for 8-15s, and extension at 68-72 deg.C for 18-25 cycles, extension at 68-72 deg.C for 25-60s,
wherein the annealing temperature T is as follows:
when Tm is more than or equal to 65 ℃, T is Tm-5 ℃;
when Tm is more than or equal to 60 ℃ and less than 65 ℃, T is 60 ℃;
when Tm is more than or equal to 55 ℃ and less than 60 ℃, T is Tm;
when Tm < 55 ℃, T is 50 ℃;
tm is the Tm of the first reverse complementary paired region.
7. The method for synthesizing DNA according to claim 6, wherein the reaction system for PCR amplification comprises the following components: DNA polymerase, reaction buffer, primer pair and dNTP,
in the reaction system, the concentration of each primer in the primer pair is 0.4-0.5 pmol/. mu.L.
8. The method for synthesizing DNA according to claim 7, wherein the reaction system for PCR amplification comprises the following components: 1 Xreaction buffer, 1-3U/. mu.L of DNA polymerase, 0.4-0.5 pmol/. mu.L of forward primer, 0.4-0.5 pmol/. mu.L of reverse primer, 0.1-0.3mM of dNTP, and water to make up the balance.
9. The method for synthesizing DNA according to any one of claims 1 to 8, further comprising a step of ligating the PCR products to each other and a step of ligating the ligation products of the PCR products to a vector,
and the connection between the PCR products is carried out by adopting a homologous recombination method.
10. The method for synthesizing DNA according to any one of claims 1 to 9, wherein the length of the DNA to be synthesized is 150-500 bp.
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