CN116426559A - Method for rapidly synthesizing target fragment from head by using long primer - Google Patents
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Abstract
Description
技术领域technical field
本发明属于合成生物学领域,具体涉及一种利用长引物快速从头合成目的片段的方法(Rapid DNA synthesis using long primers,RSLP)。The invention belongs to the field of synthetic biology, and in particular relates to a method (Rapid DNA synthesis using long primers, RSLP) for rapidly de novo synthesizing a target fragment by using long primers.
背景技术Background technique
DNA序列的化学合成为异源系统中的基因的高效表达以及基因结构功能的表征提供了一种强大的工具。然而,由于模板DNA并不总是很容易获得,化学合成使得在不使用模板DNA分子的情况下产生基因成为可能。然而基因化学合成法也存在许多缺点,包括价格高昂、合成周期长、难以合成高G+C含量、重复序列或复杂的二级结构的DNA。所以,为了避免实验周期因为目标DNA合成而变得过长,开发一种DNA简便、快速且经济的合成方法变得十分重要。The chemical synthesis of DNA sequences provides a powerful tool for the efficient expression of genes in heterologous systems and the characterization of gene structure and function. However, since template DNA is not always readily available, chemical synthesis makes it possible to generate genes without the use of template DNA molecules. However, genetic chemical synthesis also has many disadvantages, including high price, long synthesis cycle, and difficulty in synthesizing DNA with high G+C content, repetitive sequence or complex secondary structure. Therefore, in order to avoid the experimental cycle becoming too long due to target DNA synthesis, it is very important to develop a simple, fast and economical DNA synthesis method.
近年来,有些研究描述了一些基于寡核苷酸的DNA序列合成和组装方法,如基于PCR的热力学平衡由内而外合成法(TBIO)、基于双不对称性PCR两步基因合成法((DA-PCR)、重叠延伸PCR(OE-PCR)、PCR两步合成DNA法(PTDS)和基于PCR的精确合成(PAS)等等。然而,这些技术存在着明显的缺陷,例如PCR合成的错误率相对较高、PCR体系冗余、PCR程序繁琐等。In recent years, some studies have described some oligonucleotide-based DNA sequence synthesis and assembly methods, such as PCR-based thermodynamic equilibrium inside-out synthesis (TBIO), double-asymmetric PCR-based two-step gene synthesis (( DA-PCR), overlapping extension PCR (OE-PCR), PCR two-step synthetic DNA method (PTDS) and PCR-based precise synthesis (PAS), etc. However, there are obvious defects in these technologies, such as errors in PCR synthesis The rate is relatively high, the PCR system is redundant, and the PCR program is cumbersome.
发明内容Contents of the invention
发明目的:本发明所要解决的技术问题是针对现有技术的不足,提供一种利用长引物快速从头合成目的片段的方法。Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for rapidly de novo synthesis of target fragments using long primers for the deficiencies of the prior art.
为了解决上述技术问题,本发明公开了一种利用长引物快速从头合成目的片段的方法,即用于合成长而准确的DNA序列的PTDS方法的改进版本RSLP。与其他DNA PCR合成方法类似,RSLP协议的第一步是设计要合成的DNA序列;在此步骤中可以优化密码子和G+C含量,以及不良的限制酶位点或二级结构;根据DNA序列,设计并化学合成与相邻的寡核苷酸重叠约18~25bp的80~100nt寡核苷酸,即长引物。然后第二步,采用前述寡核苷酸组使用PCR产生长度约为500~1000bp的小片段DNA(图1)。此处,为了节省时间,在进行PCR的步骤时将多组体系(合成同一基因不同片段)同时进行(选取最小Tm值作为退火温度)。其中,每组中最外侧的两条引物作为正/反向外引物,使用剩余的最大偶数条内部寡核苷酸作为内引物;此步骤应使用高保真DNA聚合酶。最后第三步,将多组小片段DNA(500~1000bp)通过infusion一步克隆的方式直接组装到基因表达载体上进行基因表征(图2)。In order to solve the above technical problems, the present invention discloses a method for rapidly de novo synthesis of target fragments using long primers, that is, an improved version RSLP of the PTDS method for synthesizing long and accurate DNA sequences. Similar to other DNA PCR synthesis methods, the first step in the RSLP protocol is to design the DNA sequence to be synthesized; codons and G+C content can be optimized during this step, as well as poor restriction enzyme sites or secondary structures; according to DNA Sequence, design and chemically synthesize 80-100 nt oligonucleotides overlapping with adjacent oligonucleotides by about 18-25 bp, that is, long primers. Then in the second step, the aforementioned oligonucleotide set is used to generate small fragments of DNA with a length of about 500-1000 bp by PCR ( FIG. 1 ). Here, in order to save time, multiple sets of systems (synthesis of different fragments of the same gene) are simultaneously performed during the PCR step (choose the minimum Tm value as the annealing temperature). Among them, the two outermost primers in each group are used as forward/reverse outer primers, and the remaining largest even-numbered internal oligonucleotides are used as inner primers; high-fidelity DNA polymerase should be used for this step. Finally, in the third step, multiple groups of small fragments of DNA (500-1000 bp) were directly assembled into gene expression vectors for gene characterization by means of infusion one-step cloning (Figure 2).
所述利用长引物快速从头合成目的片段的方法具体包括如下步骤:The method for rapidly de novo synthesis of target fragments using long primers specifically includes the following steps:
(1)设计合成多条存在碱基重叠、覆盖整个目的片段的长引物;(1) Design and synthesize multiple long primers with overlapping bases and covering the entire target fragment;
(2)使用偶数条步骤(1)获得的长引物交替重叠延伸进行PCR扩增,获得PCR产物,即长度约为500~1000bp的小片段DNA;(2) Use the even-numbered long primers obtained in step (1) to carry out PCR amplification by alternate overlapping extension to obtain PCR products, that is, small fragments of DNA with a length of about 500 to 1000 bp;
(3)利用一步法同源重组将步骤(2)获得的PCR产物直接整合到线性化载体中,得到重组产物;(3) directly integrating the PCR product obtained in step (2) into a linearized vector by one-step homologous recombination to obtain a recombinant product;
(4)将步骤(3)获得的重组产物转化入感受态细胞,获得带有目的基因的转化子;(4) Transforming the recombinant product obtained in step (3) into a competent cell to obtain a transformant with the gene of interest;
所述方法从目的基因中长引物的设计到最终产品,即步骤(1)~(3)总耗时2~5h。The method takes 2-5 hours in total from the design of the long primer in the target gene to the final product, that is, steps (1)-(3).
上述方法中,若涉及退火,其温度为45℃以上,如所涉及PCR过程中,退火的温度为45℃以上,多引物PCR程序,以及载体片段线性化PCR程序中,退火的温度均为45℃以上。In the above method, if annealing is involved, the temperature is above 45°C. For example, in the PCR process involved, the annealing temperature is above 45°C. In the multi-primer PCR program and the vector fragment linearization PCR program, the annealing temperature is 45°C. ℃ or more.
步骤(1)中,根据设计的目的片段的长度选择合适的引物条数,具体为在目的基因的5’端选取80~100nt的片段作为正向外引物,在80~100nt片段的3’端获得18~25bp长的重叠片段,从第(80~100-重叠片段)bp处再向后选择80~100nt的长引物片段,以此类推到最后一个引物设计完成;所述80~100nt,优选为80~90nt,进一步优选为90nt;所述18~25bp,优选为18~22bp,进一步优选为20bp。In step (1), select the appropriate number of primers according to the length of the designed target fragment, specifically, select a fragment of 80-100nt at the 5' end of the target gene as the forward outer primer, and select a fragment at the 3' end of the 80-100nt fragment Obtain an overlapping fragment of 18-25bp long, and then select a long primer fragment of 80-100nt from the (80-100-overlapping fragment) bp, and so on until the last primer design is completed; the 80-100nt, preferably 80-90nt, more preferably 90nt; the 18-25bp, preferably 18-22bp, more preferably 20bp.
步骤(1)中,所述碱基重叠的重叠长度为18~25bp,优选为18~22bp,进一步优选为20bp。In step (1), the overlap length of the base overlap is 18-25 bp, preferably 18-22 bp, more preferably 20 bp.
步骤(1)中,所述长引物的序列长度为80~100nt,优选为80~90nt,进一步优选为90nt。DNA合成的一个重要参数是用于PCR的寡核苷酸的长度。对于RSLP协议,本发明使用90nt寡核苷酸,因为90nt在错误率和生产成本之间提供了最佳平衡。In step (1), the sequence length of the long primer is 80-100 nt, preferably 80-90 nt, more preferably 90 nt. An important parameter for DNA synthesis is the length of the oligonucleotides used for PCR. For the RSLP protocol, the present invention uses 90nt oligonucleotides because 90nt provides the best balance between error rate and production cost.
步骤(2)中,所述PCR采用无模板模式,以偶数条步骤(1)所得长引物中重叠交替延伸时最外侧的两条引物作为正/反向外引物,剩余最大偶数条的长引物作为内引物。In step (2), the PCR adopts the template-free mode, and the outermost two primers of the even-numbered long primers obtained in step (1) are used as forward/reverse outer primers when overlapping and alternately extended, and the remaining long primers with the largest even number as an internal primer.
步骤(2)中,所述PCR的体系由0.5μl终浓度为10nM内引物混合液、1μl终浓度为0.2μM正向外引物、1μl终浓度为0.2μM反向外引物、25μl高保真DNA聚合酶、dNTP以及缓冲液的混合体系,ddH2O补足至50μl组成的;优选地,所述终浓度为10nM内引物混合液为将1μl浓度为10nM内引物混匀后获得,即所述内引物均以10nM的浓度等体积混匀后获得。In step (2), the PCR system consists of 0.5 μl final concentration of 10 nM inner primer mixture, 1 μl final concentration of 0.2 μM forward outer primer, 1 μl final concentration of 0.2 μM reverse outer primer, 25 μl high-fidelity DNA polymerase A mixed system of enzyme, dNTP and buffer, made up to 50 μl of ddH 2 O; preferably, the final concentration of 10 nM internal primer mixture is obtained by mixing 1 μl of internal primer with a concentration of 10 nM, that is, the internal primer All were obtained after equal volume mixing at a concentration of 10 nM.
其中,所述DNA聚合酶和缓冲液的混合体系为Vazyme P520-01,在一些实施例中也可以高保真酶,在一些实施例中也可以使用2×Phanta Max Master Mix,可使每100,000bp<1个误差。之前所有的基因合成方法在最终产品中都存在错误,错误来源主要是寡核苷酸的突变和DNA聚合酶介导的靶DNA序列合成过程中引入的突变。Wherein, the mixed system of DNA polymerase and buffer is Vazyme P520-01, in some embodiments it can also be a high-fidelity enzyme, in some embodiments it can also use 2×Phanta Max Master Mix, which can make every 100,000bp <1 error. All previous gene synthesis methods have errors in the final product, mainly due to mutations in oligonucleotides and mutations introduced during DNA polymerase-mediated synthesis of the target DNA sequence.
步骤(3)中,所述线性化载体是通过PCR技术利用含有同源臂的正、反向引物使载体线性化得到的。In step (3), the linearized vector is obtained by using forward and reverse primers containing homology arms to linearize the vector by PCR technology.
其中,所述的含有同源臂的正、反向引物线性化之后的载体,其在5’端、3’端带有的20bp的同源臂序列分别与PCR产物5’端、3’端的序列相同。如果是多片段一起插入到线性化载体中,则是线性化载体在5’端与多个小片段组成的大片段的5’端的序列相同,3’端与多个小片段组成的大片段的3’端的序列相同。Wherein, the vector after the linearization of the forward and reverse primers containing the homology arm has a 20bp homology arm sequence at the 5' end and the 3' end of the PCR product, respectively. The sequence is the same. If multiple fragments are inserted into the linearized vector together, the sequence at the 5' end of the linearized vector is the same as that of the large fragment composed of multiple small fragments, and the sequence at the 3' end is the same as that of the large fragment composed of multiple small fragments. The sequence at the 3' end is the same.
步骤(3)中,所述一步法同源重组,其体系包括线性化载体和n个插入片段,5≥n≥1,如n=1,n=2,n=3。In step (3), the one-step homologous recombination system includes a linearized vector and n inserts, 5≥n≥1, such as n=1, n=2, n=3.
其中,所述线性化载体和n个插入片段的用量为所述的线性化载体和插入片段的摩尔比是1:1,终浓度为0.03pmol的每个线性化载体/插入片段使用量=[0.02×线性化载体/插入片段碱基对数]ng。Wherein, the amount of the linearized vector and the n inserts is that the molar ratio of the linearized vector and the insert is 1:1, and the final concentration is 0.03 pmol of each linearized vector/insert used amount=[ 0.02 × linearized vector/insert base pairs] ng.
步骤(3)中,所述一步法同源重组,其重组时间为30min,重组温度为37℃;优选地,待重组时间结束后置于4℃保存。In step (3), the one-step homologous recombination has a recombination time of 30 minutes and a recombination temperature of 37°C; preferably, it is stored at 4°C after the recombination time is over.
可见,本发明所提供的RSLP协议相对简单(多个并行的PCR后一步克隆到质粒上),快速(从目标基因的设计到最终产品~4h),精确(每100,000bp合成≤1个误差)且价格低廉。It can be seen that the RSLP protocol provided by the present invention is relatively simple (multiple parallel PCRs are cloned onto the plasmid in one step), fast (from the design of the target gene to the final product ~4h), and accurate (≤1 error per 100,000bp synthesis) And the price is low.
有益效果:Beneficial effect:
(1)相比于与本发明实验步骤类似的方案,本发明选用90nt的寡核苷酸链(长引物),既缩短了合成周期、降低了合成难度,又节约了经济成本,在错误率和生产成本之间提供了最佳的平衡。(1) Compared with the scheme similar to the experimental procedure of the present invention, the present invention selects the oligonucleotide chain (long primer) of 90nt for use, has not only shortened the synthesis period, reduced the difficulty of synthesis, but also saved economic cost, and the error rate Provides the best balance between and production costs.
(2)本发明采用了无模版,利用长的寡核苷酸链(长引物)从头合成DNA的方法,不仅在费用方面是目前市面上从头化学合成DNA链的一半以下,而且在合成高GC含量,结构复杂的DNA链方面具有无法替代的优势。最重要的是在时间消耗方面,本发明大大节约了使用者获取新基因的时间。在特定基因片段长度下,与在普通公司订购基因的周期相比,本发明成功构建基因的时耗是其的1/3倍,并且伴随着基因片段长度的增加,此发明的时间优势将被继续扩大,以合成一段10kb的DNA链为例,本发明的时耗是化学合成的1/45倍!十分适用于没有模版且亟需新基因的分子生物实验室。(2) The present invention has adopted no template, utilizes the method for de novo DNA synthesis of long oligonucleotide chains (long primers), not only in terms of cost, is less than half of the de novo chemical synthesis of DNA chains currently on the market, but also in the synthesis of high GC It has irreplaceable advantages in terms of DNA chain content and complex structure. Most importantly, in terms of time consumption, the present invention greatly saves the time for users to acquire new genes. Under the specific gene fragment length, compared with the cycle of ordering genes in ordinary companies, the time consumption of the present invention to successfully construct the gene is 1/3 times, and with the increase of the gene fragment length, the time advantage of this invention will be eliminated Continue to expand, taking the synthesis of a 10kb DNA chain as an example, the time consumption of the present invention is 1/45 times that of chemical synthesis! It is very suitable for molecular biology laboratories without templates and urgently needing new genes.
(3)本发明使用同源重组组合多基因片段,该方法将多个带有同源臂的DNA片段连接起来,可确保合成基因的保真度进一步提高,同时也能节约时间。相比于传统的PAS法连接多个小片段使用的重叠延伸PCR方法,本发明直接在引物设计之初就在相邻两个DNA片段两端加上20bp的同源臂,最后通过一步克隆的方式将人工合成的基因克隆入质粒载体中。尽管商用DNA聚合酶的保真度越来越高,但是并不能保证PCR过程绝对不会出错,特别是针对于本发明的原理。在本反应中,一定数量的酶进行的结合引物3’-OH端的次数是有模版体系的5倍以上,如果采用现有技术的PTDS法,那么在利用小片段合成大片段的过程中极易出错,这也是PTDS法面临的最大痛点之一。本发明利用同源臂的一步克隆技术可以使合成的片段插入到载体的任意位置上,且不受限于酶切位点的有无。(3) The present invention uses homologous recombination to combine multiple gene fragments. This method connects multiple DNA fragments with homologous arms, which can ensure the further improvement of the fidelity of the synthetic gene and save time. Compared with the overlap extension PCR method used by the traditional PAS method to connect multiple small fragments, the present invention directly adds 20bp homology arms to both ends of two adjacent DNA fragments at the beginning of primer design, and finally clones them in one step. Cloning of artificially synthesized genes into plasmid vectors. Despite the increasing fidelity of commercial DNA polymerases, there is no guarantee that the PCR process will be error-free, especially with regard to the principles of the present invention. In this reaction, the number of times that a certain number of enzymes bind the 3'-OH end of the primer is more than 5 times that of the template system. If the PTDS method of the prior art is used, it is very easy to synthesize large fragments using small fragments This is also one of the biggest pain points facing the PTDS law. The invention utilizes the one-step cloning technique of the homology arm to insert the synthesized fragment into any position of the vector, and is not limited by the presence or absence of restriction sites.
附图说明Description of drawings
下面结合附图和具体实施方式对本发明做更进一步的具体说明,本发明的上述和/或其他方面的优点将会变得更加清楚。The advantages of the above and/or other aspects of the present invention will become clearer as the present invention will be further described in detail in conjunction with the accompanying drawings and specific embodiments.
图1为PCR过程演示。Figure 1 is a demonstration of the PCR process.
图2为全过程流程演示图。Figure 2 is a flow chart of the whole process.
图3为pET29a-AMP质粒图谱。Fig. 3 is a plasmid map of pET29a-AMP.
图4为引物设计概念图。Figure 4 is a conceptual diagram of primer design.
图5为实施例1测序结果(部分)。Fig. 5 is the sequencing result (part) of Example 1.
图6为实施例2测序结果(部分)。Fig. 6 is the sequencing result (part) of Example 2.
具体实施方式Detailed ways
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂和材料,如无特殊说明,均可从商业途径获得。The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and materials, unless otherwise specified, can be obtained from commercial sources.
下述实施例中,所述的设计合成的长引物,其序列长度均选用90nt。因为在市面上,以金唯智公司为例,引物合成收费标准以90nt为分水岭,大于90nt的引物合成收费在4.2元/nt,小于等于90nt的引物合成收费在0.45元/nt。在合成周期及合成难度上,大于90nt的引物,其合成难度普遍比小于等于90nt引物的合成难度高,合成周期也长。而在小于等于90nt的引物合成中,以合成一段860bp长度的DNA序列为例,可以选择选择12条90nt长的引物,合成的多个引物的总长度为1080bp;也可以选择21条60nt长的引物,合成的多个引物的总长度为1260bp,但后者成本比前者高了100元。因此,在本发明的引物合成中,选用90nt作为设计合成的长引物的序列长度。In the following examples, the sequence length of the designed and synthesized long primers is all 90 nt. Because in the market, taking Jinweizhi Company as an example, the primer synthesis fee standard is 90nt as the watershed, the primer synthesis fee is 4.2 yuan/nt for primers larger than 90nt, and the primer synthesis fee for 90nt or less is 0.45 yuan/nt. In terms of synthesis cycle and synthesis difficulty, the synthesis difficulty of primers larger than 90 nt is generally higher than that of primers smaller than or equal to 90 nt, and the synthesis cycle is also longer. In the synthesis of primers less than or equal to 90nt, taking the synthesis of a DNA sequence with a length of 860bp as an example, you can choose 12 primers with a length of 90nt, and the total length of multiple primers synthesized is 1080bp; you can also choose 21 primers with a length of 60nt. Primers, the total length of multiple synthetic primers is 1260bp, but the cost of the latter is 100 yuan higher than that of the former. Therefore, in the primer synthesis of the present invention, 90nt is selected as the sequence length of the long primer designed and synthesized.
下述实施例1和实施例2中PCR的最大耗时为1.5h,也就是1.5h内可完成PCR操作。The maximum time-consuming of PCR in the following examples 1 and 2 is 1.5h, that is, the PCR operation can be completed within 1.5h.
实施例1:pET29a-EI-AMP质粒的构建Embodiment 1: Construction of pET29a-EI-AMP plasmid
获取目的基因序列EI【BBa_K1317003】,对其进行密码子优化使之适用于Escherichia coli,优化后的序列如SEQ ID NO.3所示。使用snapgene软件导入目的基因并设计合成所用的长引物。首先在目的基因的5’端选取90nt的片段作为正向外引物,在90nt片段的3’端获得20bp长的重叠片段,从第(90-重叠片段)bp处再向后选择90nt的长引物片段,以此类推到最后一个引物设计完成。注意:所设计的每条引物延伸方向须与上一条相反。保证设计的引物总数为偶数(引物设计直观展示见图4)。长引物序列如SEQ ID NO.4-NO.15所示。耗时30min。The target gene sequence EI [BBa_K1317003] was obtained, and its codon optimization was performed to make it suitable for Escherichia coli. The optimized sequence is shown in SEQ ID NO.3. Use the snapgene software to import the target gene and design the long primers used for synthesis. First, select a 90nt fragment at the 5' end of the target gene as a forward primer, obtain a 20bp long overlapping fragment at the 3' end of the 90nt fragment, and then select a 90nt long primer backward from the (90-overlapping fragment) bp Fragments, and so on until the last primer design is completed. Note: The extension direction of each designed primer must be opposite to that of the previous one. Ensure that the total number of primers designed is an even number (see Figure 4 for a visual display of primer design). The long primer sequences are shown in SEQ ID NO.4-NO.15. It takes 30 minutes.
使用如下步骤配置体系并进行PCR扩增反应:Use the following steps to configure the system and perform PCR amplification reactions:
一、多引物PCR:合成一段855bp的DNA链。耗时:1h5min。1. Multi-primer PCR: Synthesize a 855bp DNA strand. Time-consuming: 1h5min.
(1)将设计合成的90nt寡核苷酸长引物组成1组,其中包含的12个寡核苷酸长引物(内引物:SEQ ID NO.5-14、正向外引物:SEQ ID NO.4,反向外引物:SEQ ID NO.15)需要合成一个855bp的DNA片段。在整个过程中,除非另有说明,否则所有反应溶液都应在冰上储存和处理。(1) The designed and synthesized 90nt long oligonucleotide primers were composed into a group, which contained 12 long oligonucleotide primers (inner primers: SEQ ID NO.5-14, forward outer primers: SEQ ID NO. 4. The reverse outer primer: SEQ ID NO.15) needs to synthesize a 855bp DNA fragment. Throughout the procedure, all reaction solutions should be stored and handled on ice unless otherwise stated.
(2)将每组10个内引物(除去最外侧正/反向外引物的其他引物)进行混合:分别取1μl(10μM)10个内引物置于一个200μl的PCR管中。通过移液枪混合均匀并用离心机短暂离心将所有液体置于管底。(2)
(3)取0.5μl第(2)步的内引物混合溶液置于200μl的PCR管中,再分别取1μl(10nM)正向外引物和反向外引物置于上述200μl的PCR管中。每个内引物终浓度控制在10nM,外引物终浓度控制在0.2μM。多引物PCR体系如表1所示。(3) Take 0.5 μl of the inner primer mixture solution in step (2) and place it in a 200 μl PCR tube, and then take 1 μl (10 nM) of the forward outer primer and reverse outer primer respectively and place them in the above-mentioned 200 μl PCR tube. The final concentration of each internal primer was controlled at 10 nM, and the final concentration of the external primer was controlled at 0.2 μM. The multi-primer PCR system is shown in Table 1.
表1多引物PCR体系设置Table 1 Multi-primer PCR system setup
(4)使用表2所示的PCR程序(4) Use the PCR program shown in Table 2
表2多引物PCR程序设置Table 2 Multi-primer PCR program settings
(5)取5μl的PCR产物加入到1.0%的含有0.1%的核酸染料的琼脂糖凝胶中电泳30min,电压为125V,使用DL5000 DNA marker(MD102-01,Vazyme)作为参照。(5) 5 μl of the PCR product was added to 1.0% agarose gel containing 0.1% nucleic acid dye for electrophoresis for 30 min at a voltage of 125 V, using DL5000 DNA marker (MD102-01, Vazyme) as a reference.
(6)质粒pET29a-AMP来源于实验室留存,由金斯瑞生物科技股份有限公司合成。(图3),设计含有同源臂的引物(正向引物核苷酸序列如SEQ ID NO.1所示,反向引物核苷酸序列如SEQ ID NO.2所示),通过PCR的方式使其线性化。PCR体系以及程序如表3和表4所示。(6) Plasmid pET29a-AMP is derived from laboratory preservation and synthesized by GenScript Biotechnology Co., Ltd. (Fig. 3), design the primer (forward primer nucleotide sequence as shown in SEQ ID NO.1, reverse primer nucleotide sequence as shown in SEQ ID NO.2) that contains homology arm, by the mode of PCR to make it linear. The PCR system and program are shown in Table 3 and Table 4.
表3载体片段线性化PCR体系Table 3 Vector fragment linearization PCR system
表4载体片段线性化PCR程序Table 4 Vector fragment linearization PCR program
二、回收目的条带DNA。耗时:15min。2. Recover the DNA of the target band. Time-consuming: 15 minutes.
(1)使用核酸胶切割设备将目的条带切割下来,使用FastPure Gel DNAExtraction Mini Kit Vazyme进行胶回收。(1) Use the nucleic acid gel cutting equipment to cut off the target band, and use the FastPure Gel DNAExtraction Mini Kit Vazyme for gel recovery.
(2)使用超微量紫外分光光度计对胶回收产物进行定量(注意,胶回收的DNA片段浓度至少在20ng/μl以上)。(2) Use an ultra-micro-volume ultraviolet spectrophotometer to quantify the products recovered from the gel (note that the concentration of DNA fragments recovered from the gel should be at least 20 ng/μl).
三、一步克隆。耗时:30min.Three, one-step cloning. Time-consuming: 30min.
(1)按照表5配置一步克隆体系:(1) Configure the one-step cloning system according to Table 5:
表5一步克隆体系Table 5 One-step cloning system
注:线性化载体和插入片段的摩尔比是1:1,线性化载体以及插入片段量化公式:终浓度为0.03pmol的每个线性化载体/插入片段最适使用量=[0.02×线性化载体/插入片段碱基对数]ng,这点对于重组是否成功十分重要;n=1。Note: The molar ratio of linearized vector and insert is 1:1, linearized vector and insert quantification formula: the optimal amount of each linearized vector/insert with a final concentration of 0.03pmol=[0.02×linearized vector /number of base pairs of the insert] ng, which is very important for the success of the recombination; n=1.
(2)重组时间严格控制在30min,重组温度为37℃,之后立即放在4℃保存,获得重组产物pET29a-EI-AMP。重组产物放在-20℃可存放一周,待需要时解冻即可。(2) The recombination time was strictly controlled at 30 min, the recombination temperature was 37°C, and immediately stored at 4°C to obtain the recombination product pET29a-EI-AMP. The recombinant product can be stored at -20°C for one week, and can be thawed when needed.
四、重组产物的转化4. Transformation of recombinant products
(1)在冰上解冻克隆用的化学感受态细胞(DH5a Competent cell,Vazyme C502)。(1) Thaw chemically competent cells (DH5a Competent cell, Vazyme C502) for cloning on ice.
(2)取10μl重组产物加入到100μl感受态细胞中,轻弹管壁混匀(请勿振莎混匀),冰上静置30min。(2) Take 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix (do not vibrate to mix), and let stand on ice for 30 minutes.
(3)42℃水浴热激45sec后,立即置于冰上冷却2-3min。(3) After heat-shocking in a water bath at 42°C for 45 sec, immediately place it on ice to cool for 2-3 min.
(4)加入900μl SOC或LB培养基(不添加抗生素),37’C摇菌1h(转速200-250rpm)。(4) Add 900 μl of SOC or LB medium (without adding antibiotics), and shake the bacteria at 37'C for 1 hour (rotating speed 200-250rpm).
(5)将含有终浓度为100mg/L卡纳霉素抗性的LB平板固体培养基在37℃培养箱中预热。(5) Preheat the LB plate solid medium containing kanamycin resistance at a final concentration of 100 mg/L in a 37° C. incubator.
(6)5,000rpm(2400×g)离心5min,弃掉900μl上清。用剩余SOC或LB培养基将菌体重悬,用无菌涂布棒在含有正确抗性的平板上轻轻涂匀。(6) Centrifuge at 5,000 rpm (2400×g) for 5 minutes, and discard 900 μl of supernatant. Resuspend the bacteria with the remaining SOC or LB medium, and spread evenly on the plate containing the correct resistance with a sterile spreader stick.
(7)37℃培养箱中倒置培养12-16h。(7) Incubate upside down in a 37°C incubator for 12-16 hours.
五、验证Five, verification
挑取转化成功的菌落,利用菌落PCR验证,将菌落PCR阳性转化子进行接种,扩大培养,保藏,提取质粒,进行质粒pET29a-EI-AMP的PCR验证。对于验证结果呈阳性的转化子,委托苏州金唯智生物科技有限公司进行测序。测序结果正确的即确认为重组菌(测序结果见图5)。Pick the successfully transformed colonies, use colony PCR verification, inoculate the colony PCR positive transformants, expand culture, preserve, extract the plasmid, and carry out the PCR verification of the plasmid pET29a-EI-AMP. For transformants with positive verification results, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to perform sequencing. If the sequencing result is correct, it is confirmed as the recombinant bacterium (see Figure 5 for the sequencing result).
实施例2:pET29a-OVA质粒的构建Embodiment 2: Construction of pET29a-OVA plasmid
获取目的基因序列OVA【BBa_K4150006】,对其进行密码子优化使之适用于Escherichia coli,优化后的序列如SEQ ID NO.16所示。使用snapgene软件导入目的基因并设计合成所用的长引物,首先在目的基因的5’端选取90nt的片段作为正向外引物,在90nt片段的3’端获得20bp长的重叠片段,从第(90-重叠片段)bp处再向后选择90nt的长引物片段,以此类推到最后一个引物设计完成。注意:所设计的每条引物延伸方向须与上一条相反。保证设计的引物总数为偶数(引物设计直观展示见图4)。长引物序列如SEQ IDNO.17-NO.34所示。耗时40min。Obtain the target gene sequence OVA [BBa_K4150006], and optimize its codons to make it suitable for Escherichia coli. The optimized sequence is shown in SEQ ID NO.16. Use the snapgene software to import the target gene and design the long primers used for synthesis. First, a 90nt fragment is selected at the 5' end of the target gene as a forward primer, and a 20bp long overlapping fragment is obtained at the 3' end of the 90nt fragment. From (90 -overlapping fragment) bp and then select a 90nt long primer fragment backwards, and so on until the last primer design is completed. Note: The extension direction of each designed primer must be opposite to that of the previous one. Ensure that the total number of primers designed is an even number (see Figure 4 for a visual display of primer design). The long primer sequences are shown in SEQ ID NO.17-NO.34. It takes 40 minutes.
使用如下步骤配置体系并进行PCR扩增反应:Use the following steps to configure the system and perform PCR amplification reactions:
一、多引物PCR:合成两段长度分别是571bp和677bp的DNA链。耗时:1.5h。1. Multi-primer PCR: synthesize two DNA strands with lengths of 571bp and 677bp respectively. Time-consuming: 1.5h.
(1)将所有设计合成的90nt寡核苷酸长引物分成适当数量的2组,其中第一组8个寡核苷酸引物(内引物:SEQ ID NO.18-23、正向外引物:SEQ ID NO.17,反向外引物:SEQ IDNO.24)需要合成一个571bp的DNA片段,第二组10个寡核苷酸引物(内引物:SEQ ID NO.26-33、正向外引物:SEQ ID NO.25、反向外引物:SEQ ID NO.34)需要合成一个677bp的DNA片段。在整个过程中,除非另有说明,否则所有反应溶液都应在冰上储存和处理。(1) Divide all designed and synthesized 90nt oligonucleotide long primers into 2 groups of appropriate numbers, wherein the first group of 8 oligonucleotide primers (inner primers: SEQ ID NO.18-23, forward outer primers: SEQ ID NO.17, reverse outer primer: SEQ ID NO.24) need to synthesize a 571bp DNA fragment, the second group of 10 oligonucleotide primers (inner primer: SEQ ID NO.26-33, forward outer primer : SEQ ID NO.25, reverse outer primer: SEQ ID NO.34) need to synthesize a 677bp DNA fragment. Throughout the procedure, all reaction solutions should be stored and handled on ice unless otherwise stated.
(2)将每组的内引物进行预混合:分别取两组1μl(10μM)内引物分别置于两个200μl的PCR管中。通过移液枪混合均匀并用离心机短暂离心将所有液体置于管底。(2) Premix the internal primers of each group: take two groups of 1 μl (10 μM) internal primers and place them in two 200 μl PCR tubes. Mix well by pipetting and centrifuge briefly to bring all liquid to the bottom of the tube.
(3)分别取0.5μl第(2)步的两组内引物混合溶液置于200μl的PCR管中,再分别取1μl(10nM)每组对应的正向外引物和反向外引物置于上述200μl的PCR管中。每个内引物终浓度控制在10nM,外引物终浓度控制在0.2μM。多引物PCR体系如表6所示。(3) Take 0.5 μl of the two groups of internal primer mixture solutions in step (2) and place them in a 200 μl PCR tube, and then take 1 μl (10 nM) of each group’s corresponding forward and reverse external primers and place them in the above 200μl PCR tube. The final concentration of each internal primer was controlled at 10 nM, and the final concentration of the external primer was controlled at 0.2 μM. The multi-primer PCR system is shown in Table 6.
表6多引物PCR体系设置Table 6 Multi-primer PCR system setup
(4)使用表7所示的PCR程序(4) Use the PCR program shown in Table 7
表7多引物PCR程序设置Table 7 Multi-primer PCR program setting
(5)分别取5μl的PCR产物加入到1.0%的含有0.1%的核酸染料的琼脂糖凝胶中电泳30min,电压为125V,使用DL5000 DNA marker(MD102-01,Vazyme)作为参照。(5) 5 μl of PCR products were added to 1.0% agarose gel containing 0.1% nucleic acid dye for electrophoresis for 30 min at a voltage of 125 V, using DL5000 DNA marker (MD102-01, Vazyme) as a reference.
(6)质粒pET29a-AMP来源于实验室留存,由金斯瑞生物科技股份有限公司合成。(图3),设计含有同源臂的引物(正向引物核苷酸序列如SEQ ID NO.35所示,反向引物核苷酸序列如SEQ ID NO.36所示),通过PCR的方式使其线性化。PCR体系以及程序如表8和表9所示。(6) Plasmid pET29a-AMP is derived from laboratory preservation and synthesized by GenScript Biotechnology Co., Ltd. (Fig. 3), design the primer (forward primer nucleotide sequence as shown in SEQ ID NO.35, reverse primer nucleotide sequence as shown in SEQ ID NO.36) that contains homology arm, by the mode of PCR to make it linear. The PCR system and program are shown in Table 8 and Table 9.
表8载体片段线性化PCR体系Table 8 Vector fragment linearization PCR system
表9载体片段线性化PCR程序Table 9 Vector fragment linearization PCR program
二、回收目的条带DNA。耗时:15min。2. Recover the DNA of the target band. Time-consuming: 15 minutes.
(1)使用核酸胶切割设备将目的条带切割下来,使用FastPure Gel DNAExtraction Mini Kit Vazyme进行胶回收。(1) Use the nucleic acid gel cutting equipment to cut off the target band, and use the FastPure Gel DNAExtraction Mini Kit Vazyme for gel recovery.
(2)使用超微量紫外分光光度计对胶回收产物进行定量(注意,胶回收的DNA片段浓度至少在20ng/μl以上)。(2) Use an ultra-micro-volume ultraviolet spectrophotometer to quantify the products recovered from the gel (note that the concentration of DNA fragments recovered from the gel should be at least 20 ng/μl).
四、一步克隆。耗时:30min.Four, one-step cloning. Time-consuming: 30min.
(1)按照表10配置一步克隆体系:(1) Configure the one-step cloning system according to Table 10:
表10一步克隆体系Table 10 One-step cloning system
注:线性化载体和插入片段的摩尔比是1:1,线性化载体以及插入片段量化公式:终浓度为0.03pmol的每个线性化载体/插入片段最适使用量=[0.02×线性化载体/插入片段碱基对数]ng,这点对于重组是否成功十分重要;n=2。Note: The molar ratio of linearized vector and insert is 1:1, linearized vector and insert quantification formula: the optimal amount of each linearized vector/insert with a final concentration of 0.03pmol=[0.02×linearized vector /number of base pairs of the insert] ng, which is very important for the success of the recombination; n=2.
(2)重组时间严格控制在30min,重组温度为37℃,之后立即放在4℃保存,获得重组产物pET29a-OVA。重组产物放在-20℃可存放一周,待需要时解冻即可。(2) The recombination time was strictly controlled at 30 minutes, the recombination temperature was 37°C, and immediately stored at 4°C to obtain the recombination product pET29a-OVA. The recombinant product can be stored at -20°C for one week, and can be thawed when needed.
四、重组产物的转化4. Transformation of recombinant products
(1)在冰上解冻克隆用的化学感受态细胞(DH5a Competent cell,Vazyme C502)。(1) Thaw chemically competent cells (DH5a Competent cell, Vazyme C502) for cloning on ice.
(2)取10μl重组产物加入到100μl感受态细胞中,轻弹管壁混匀(请勿振莎混匀),冰上静置30min。(2) Take 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix (do not vibrate to mix), and let stand on ice for 30 minutes.
(3)42℃水浴热激45sec后,立即置于冰上冷却2-3min。(3) After heat-shocking in a water bath at 42°C for 45 sec, immediately place it on ice to cool for 2-3 min.
(4)加入900μl SOC或LB培养基(不添加抗生素),37’C摇菌1h(转速200-250rpm)。(4) Add 900 μl of SOC or LB medium (without adding antibiotics), and shake the bacteria at 37'C for 1 hour (rotating speed 200-250rpm).
(5)将含有终浓度为100mg/L卡纳霉素抗性的LB平板固体培养基在37℃培养箱中预热。(5) Preheat the LB plate solid medium containing kanamycin resistance at a final concentration of 100 mg/L in a 37° C. incubator.
(6)5,000rpm(2400×g)离心5min,弃掉900μl上清。用剩余SOC或LB培养基将菌体重悬,用无菌涂布棒在含有正确抗性的平板上轻轻涂匀。(6) Centrifuge at 5,000 rpm (2400×g) for 5 minutes, and discard 900 μl of supernatant. Resuspend the bacteria with the remaining SOC or LB medium, and spread evenly on the plate containing the correct resistance with a sterile spreader stick.
(7)37℃培养箱中倒置培养12-16h。(7) Incubate upside down in a 37°C incubator for 12-16 hours.
五、验证Five, verification
挑取转化成功的菌落,利用菌落PCR验证,将菌落PCR阳性转化子进行接种,扩大培养,保藏,提取质粒,进行质粒pET29a-OVA的PCR验证。对于验证结果呈阳性的转化子,委托苏州金唯智生物科技有限公司进行测序。测序结果正确的即确认为重组菌(测序结果见图6)。Pick the successfully transformed colonies, use colony PCR verification, inoculate the colony PCR positive transformants, expand culture, preserve, extract the plasmid, and carry out the PCR verification of the plasmid pET29a-OVA. For transformants with positive verification results, entrust Suzhou Jinweizhi Biotechnology Co., Ltd. to perform sequencing. If the sequencing result is correct, it is confirmed as the recombinant bacterium (see Figure 6 for the sequencing result).
综合全套实验流程,本发明改进的利用长引物从头合成DNA的方法能够高效、高保真得合成理性设计的多个DNA片段,实验周期(从设计基因到构建成功重组质粒)相比于市面上公司里常规的DNA合成,时间减少了至少5天以上。Integrating a complete set of experimental procedures, the improved method of de novo DNA synthesis using long primers in the present invention can synthesize rationally designed multiple DNA fragments with high efficiency and high fidelity. For conventional DNA synthesis, the time is reduced by at least 5 days.
本发明提供了一种利用长引物快速从头合成目的片段的方法的思路及方法,具体实现该技术方案的方法和途径很多,以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。The present invention provides an idea and method for using long primers to rapidly de novo synthesize target fragments. There are many methods and approaches to realize this technical solution. The above descriptions are only preferred embodiments of the present invention. Those of ordinary skill in the art can make some improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. All components that are not specified in this embodiment can be realized by existing technologies.
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