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Published: 17 March 2017

A restriction-costless method for gene reconstitution using two single-primer PCRs in parallel to generate compatible cohesive ends

BMC Biotechnology volume 17, Article number:32 (2017) Cite this commodity

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Abstract

Background

Restriction-costless (RF) cloning, a PCR-based method for the creation of custom DNA plasmids, allows for the insertion of any sequence into any plasmid vector at any desired position, independent of restriction sites and/or ligation. Here, we draw a simple and fast method for performing gene reconstitution past modified RF cloning.

Results

Double-stranded inserts and acceptors were offset amplified past regular PCR. The amplified fragments were then used as the templates in two separate linear amplification reactions containing either forward or reverse primer to generate two single-strand contrary-complement counterparts, which could amalgamate to each other. The annealed inserts and acceptors with 5' and 3' cohesive ends were sealed by ligation reaction. Using this method, we fabricated 46 constructs containing insertions of upward to 20 kb. The average cloning efficiency was higher than 85%, as confirmed by colony PCR and sequencing of the inserts.

Conclusions

Our method provides an culling cloning method capable of inserting any Deoxyribonucleic acid fragment of up to at least 20 kb into a plasmid, with high efficiency. This new method does not require brake sites or alterations of the plasmid or the cistron of involvement, or boosted treatments. The simplicity of both primer blueprint and the procedure itself makes the method suitable for high-throughput cloning and structural genomics.

Background

The manipulation of recombinant Deoxyribonucleic acid molecules is an indispensable pace in modernistic high-throughput protein crystallization studies [1]. Restriction enzyme/ligase cloning, which relies on restriction enzyme digestion and ligation, is a uncomplicated and easy style to move a fragment of double-stranded DNA from one plasmid to another [2]. However, this technique has ii limitations [3]: it is ineffective when lack of unique restriction sites and sometimes results in introduction of unwanted actress sequences. To circumvent these limitations, various restriction endonuclease cleavage site–contained cloning methods have recently been adult [iv–21]. These methods have made cloning more than accessible in cases in which conventional restriction site cloning was difficult or impossible. While equally alternative cloning strategies are still required for more choices.

The brake-gratuitous (RF) cloning strategy, as described extensively in the literature, was developed as a powerful tool for reconstituting genes in circular vectors [iii, 22]. Because RF cloning requires no alterations in the plasmid or the gene of interest, it is uncommonly well-suited for high-throughput cloning. The cistron of interest is amplified in a regular polymerase concatenation reaction (PCR), which produces a primer pair that, in one case annealed to the vector of interest, is extended in a linear amplification reaction. Thus, this method relies on amplified genes functioning as primers. However, this approach also has limitations. First, the motion of big Dna fragments and the formation of secondary structures will affect the efficiency of PCR. 2nd, this method relies on digestion with DpnI, which cleaves methylated DNA, to remove parental plasmids. The efficiency of DpnI treatment is influenced by many factors, and requires vector propagation in Dam⁺ strains [23].

In this paper, we describe a simple and fast method for performing gene reconstitution by modified brake-complimentary (MRF) cloning. In this method, two rounds of PCR generate two DNA fragments with compatible v' or 3' cohesive ends, which are therefore able to ligate to each other. This new method is independent of the being of restrictions sites and DpnI treatment. Using this method, we made 46 constructs with inserts of variable size, with average cloning efficiency college than 85%. The efficiency was not significantly affected by the insert length up to 20 kb.

Results

Method overview and primer pattern

Effigy one shows the scheme for MRF cloning. Nosotros ascertain the 5–8 bp Deoxyribonucleic acid fragments earlier the insert site A as "5' overhang" and those after insert site B as "3' overhang" (see Fig. 1, Additional file one: Figure S1 and Additional file 2: Figure S2). To supplant a gene (Fig. 1, crimson line) in a vector between sites A and B, we designed eight primers (Boosted file iii: Table S1): primer 1, frontward primer, which contains a ~25 bp sequence homologous to the positive strand of the gene; primer 2, reverse primer, which contains a ~25 bp sequence homologous to the negative strand of the gene; primer 3, frontwards primer, which is the aforementioned as primer 1 but has an boosted 5' overhang at the 5' end; primer 4, contrary primer, which is the same as primer 2 simply has an additional iii' overhang at the 5' terminate; primer v, forward primer, which contains a ~25 bp sequence homologous to the negative strand of the vector; primer vi, reverse primer, which contains a ~25 bp sequence homologous to the positive strand of the vector; primer 7, contrary primer, which is the same as primer v but has an additional 5' overhang at the 5' end; primer 8, opposite primer, which is the same as primer 6 but has an boosted 3' overhang at the five' stop. All primers were designed with G or C as the 5' and three' terminal nucleotide.

Nosotros used two pairs of primers, primer one/primer 2 and primer five/primer 6, to generate two Deoxyribonucleic acid fragments, using the target factor and vector as templates, respectively. The resultant PCR products were gel purified. Nosotros and then amplified these two Dna fragments in two separate PCR reactions containing either forward or contrary primer, which will add five' overhang or 3' overhang to the PCR products (Fig. ane). Finally, Deoxyribonucleic acid fragments with complementary overhang at the v' or 3' end were able to anneal to each other, and were joined past Dna ligase (Fig. 1). The ligated products were and then transformed into DH5α competent cells. The inserted genes were verified by colony PCR and further confirmed by DNA sequencing.

MRF cloning can gather insert Dna fragment into target vector

We first tested this protocol to reconstitute the E. coli radA gene into pET22b. Based on the initial success of the protocol, we continued to employ it to generate the constructs needed for our studies. For example, we planned to replace the radA gene in vector pET22b betwixt the start codon ATG (289) and the sequence CACCACCACCACCACCAC (157) (Fig. 1 and Additional file 1: Figure S1) to yield a new construct with the gene under the control of a T7 promoter and a C-terminal His₆-tag to facilitate protein purification. Every bit shown in Fig. 1, to replace the radA gene in the vector, ii parallel PCRs were performed to dilate each Deoxyribonucleic acid fragment using the primer pairs pet22b1/pet22b2 and radA1/radA2, equally shown in Additional file three: Table S1, using pET22b or Eastward. coli genomic DNA as templates to generate Deoxyribonucleic acid fragments "i" and "2". Amplified products were separated by i% agarose gel electrophoresis and purified by gel extraction. We then performed ii carve up single-primer linear PCRs: (1) using pet22b3 or pet22b4 alone, with Dna fragment "1" as the template, to obtain single-strand DNA fragment "3-ane" or "3-2"; and (2) using radA3 or radA4 alone, with Dna fragment "2" as template, to obtain single-strand Deoxyribonucleic acid fragment "4-1" or "4-2". Fragments "3-1" and "three-2" and "iv-1" and "4-two" were then annealed to obtain double-strand DNA fragments "3" and "four", which take glutinous ends that tin ligate with other compatible ends. For single-primer PCR, we used ~500 ng of template, about 10 times more than the standard corporeality recommended for double-primer PCR (Table ane), every bit Deoxyribonucleic acid amplification in single-primer PCR is linear (merely thirty-fold for 30 PCR cycles). As the efficiency of cohesive ligation is college than that of blunt-stop ligation, the parental DNA of the products of 2d-round PCR did not need to be removed. After PCR purification, these second-round PCR products were ready for ligation. In each transformation, nosotros routinely checked eight colonies at random from each transformation past colony PCR with a forward primer annealing to vector and a opposite primer annealing to the inserted cistron.

Table i PCR reaction components

Full size table

The agarose gel in Fig. 2 shows the Dna products of one sample at successive steps of our procedure. Plasmid lonely, prior to PCR, shows two major bands (Fig. 2, lane 1). After the double-primer PCR with forward (F) and reverse (R) primers, major bands can exist seen at the expected sizes corresponding to the PCR-synthesized linear DNA (Fig. 2, lanes 2 and 3). Single-primer PCR with forwards (F) or contrary (R) yielded bands at the expected size of ~v.5 kb (Fig. 2, lane v) and ~one.4 kb (Fig. 2, lane vi) later on annealing the products of single-primer PCR, representing the PCR-synthesized linear Deoxyribonucleic acid with cohesive ends. Boosted smaller bands represent non-specific PCR products or single-strand Dna. The ligation of insert into plasmid vector is performed past T4 Deoxyribonucleic acid ligase using a molar ratio of 1:3 vector to insert. As shown in Fig. two, lane 8 (before ligation) and 9 (afterward ligation), insert and vector were ligated to ane another and shifted to a higher molecular weight. The inserted genes were amplified by colony PCR. The presence of forrard and reverse cloning sites were confirmed past Deoxyribonucleic acid sequencing (Fig. 3a).

Long Dna fragment cloning

To test the suitability of our method for a big Deoxyribonucleic acid fragment, we used it to insert a xx kb DNA fragment from the E. coli genome (200485-220925, Factor Cluster 3, Table 2 and Additional file 4: Table S4) containing 21 genes into pET22b between the outset codon ATG (289), and the sequence CACCACCACCACCACCAC (157) (Boosted file 1: Effigy S1). Gene Cluster three was amplified using the primer pair GeneCluster3-ane/GeneCluster3-ii, equally shown in Boosted file 4: Tabular array S4, using E. coli genomic Deoxyribonucleic acid equally template (Fig. two, lane 4). Gene Cluster iii with a sticky end was generated by single-primer PCR, as shown in Fig. 1, using primer GeneCluster3-three or GeneCluster3-4 (Additional file 4: Table S4) (Fig. 2, lane 7). The ligation of Gene Cluster 3 Deoxyribonucleic acid fragment into pET22b was performed with T4 DNA ligase using a tooth ratio of vector to insert at 6:i. As shown in Fig. 2, lane 10 (before ligation) and eleven (afterwards ligation), insert and vector were ligated to 1 another and shifted to a higher molecular weight. The inserted genes were amplified by colony PCR. The presence of forward and opposite cloning sites were confirmed past DNA sequencing (Fig. 3b).

Table two Genes cloned and efficiency of reconstitution

Total size table

Application of MRF cloning in genes reconstitution

In routine awarding of our cloning method, we created 46 constructs from East. coli genomic Deoxyribonucleic acid and human being cDNA (Clontech) with inserts of variable size (Table two). E. coli genes and gene clusters (Additional file 5: Tabular array S2, Additional file 6: Table S3, and Additional file 4: Table S4) were cloned from the E. coli genome. The E. coli genes and gene clusters were subcloned into pET22b, with the cistron nether the control of a T7 promoter, and with a C-terminal His₆-tag to facilitate protein purification (Additional file i: Effigy S1). Human genes were subcloned into the expression vector pcDNA™ three.ane (+) (Invitrogen) (Additional file ii: Effigy S2). Under our test weather condition, we achieved an average cloning efficiency of 86.9%. DNA sequencing revealed that all genes were correctly placed in the plasmid.

Discussion

In this study, nosotros describe a new cloning method. The technique uses two rounds of PCR to obtain inserts and acceptors with uniform cohesive ends, which are and then ligated. Using this method, we fabricated 46 constructs with inserts of variable size. The average cloning efficiency was 86.9%, as determined by colony PCR and sequencing of the cloned genes. Because the method relies on PCR to generate cohesive v' or 3' ends for DNA ligation, restriction sites are not needed, which facilitates cloning of the factor of interest. For convenience, we only used the vector pET22b for Eastward. coli genes and pcDNA™ 3.one (+) for man genes, but used inserts of variable size. Our results showed that cloning efficiency was not significantly afflicted by the unlike inserts, thus providing a glimpse of the broad choice in inserts that can be used as a template, which then tin can be used as an alternative method for multiple fragment assembly and library structure.

Nosotros noticed that cloning efficiency was not contradistinct dramatically by fragment length. Equally shown in Table ii and Additional file 7: Effigy S3, this method was suitable for the cloning of big Dna sequences upwardly to xx kb in size. In contrast to traditional restriction enzyme cloning, the method described here provides a much more flexible arroyo to gene cloning. Therefore, it represents a toll-effective and elementary solution for high-throughput cloning applications. Because this method relies on PCR distension of the Dna sequences, the virtually crucial requirement is high-fidelity DNA polymerase. Fortunately, the high-fidelity polymerases recently developed for cloning, east.1000., Phusion^® Loftier-Fidelity DNA Polymerase and KOD Hot Start Deoxyribonucleic acid Polymerase, take extremely depression error rates. Therefore, it is no longer challenging to amplify large Dna fragments for use in our method.

Conclusions

We developed a novel cloning method that provides an culling approach to DNA assembly. This method is independent of restriction sites and DpnI treatment, and does not innovate undesired operational sequences at the junctions of functional modules. This new method simplifies circuitous cloning procedures in which long stretches of Deoxyribonucleic acid can be inserted into circular plasmids in an unrestricted way, and the efficiency does not decrease for long inserts up to 20 kb. The simplicity of both primer design and the procedure itself makes the method suitable for high-throughput studies. The protein of interest is expressed without the addition of extra residues originating from the cloning procedure, making it an attractive culling method for structural genomics.

Methods

Materials

Phusion® High-Fidelity Dna Polymerase, Dna marker, Taq DNA polymerase, and T4 DNA ligase were purchased from New England Biolabs, and cloning kits from Qiagen. pET22b, pcDNA™ iii.1 (+), and host strain Escherichia coli DH5α were obtained from Invitrogen. Man cDNAs were purchased from Clontech. Oligonucleotide primers were purchased from Invitrogen. PCR purification and gel extraction kits were purchased from Qiagen. Plasmids were isolated using a QIAprep Spin Miniprep Kit (Qiagen). All other chemicals used in the study were of molecular biological science grade.

Touchdown PCR

PCR reactions were performed to generate DNA fragments in a final volume of 50 μL using Phusion® High-Fidelity Deoxyribonucleic acid Polymerase (New England Biolabs) and the primer pair every bit shown in Boosted file 3: Table S1. After the initial denaturation pace at 98 °C for 5 min, the PCR was conducted for 20 cycles with denaturation at 98 °C for twenty s; primer annealing from threescore °C to 50 °C with a step of -0.5 °C each wheel for xx southward; extension at 72 °C for thirty due south/kb; and 10 cycles with an annealing temperature at 52 °C. When all cycles were completed, the samples were maintained at 72 °C for ten min to stop all Dna synthesis.

Ligation

DNA ligation reactions were performed to fuse DNA fragments in a final volume of 20 μL using T4 Dna ligase (New England Biolabs) post-obit the standard protocol from New England Biolabs. In brief, the longer and shorter Deoxyribonucleic acid fragments were mixed at a molar ratio of one:3–1:10. The reaction was incubated at room temperature for 2 h. After rut inactivation at 65 °C for 10 min, the reaction was chilled on ice. A 10 μL aliquot of the reaction was used to transform 50 μL of competent cells.

Colony PCR

For each transformation, eight colonies were selected randomly for colony PCR to verify insertion. The colony PCR included 5 units of Taq Deoxyribonucleic acid polymerase (New England Biolabs) and 1× ThermoPol® Buffer (New England Biolabs) in the presence of 200 μM dNTP, 1 mmol each of a primer from the vector and a primer from the insert gene, and a small corporeality of cells picked from the colony, all in a last volume of xx μL. The colony PCR conditions were as follows: 95 °C for two min; 25 cycles of 95 °C for 30 s, l °C for 30 due south, and 68 °C for 1 min/kb; and a terminal extension at 68 °C for x min. Insert-positive constructs were confirmed by DNA sequencing.

Isolation and purification of total genomic DNA from E. coli

Mid-log stage E. coli DH5α cells were collected past centrifugation at four °C for ten min. The pellet was resuspended in 190 μL of TE supplemented with 10 μL of x% SDS and ane μL of twenty mg/mL protease Thou, and then incubated at 37 °C for 1 h. Subsequently 1 h, 30 μL of v M NaCl and 30 μL of CTAB/NaCl were added to the solution, and the sample was incubated at 65 °C for 20 min. Later incubation, 300 μL of phenol/chloroform/isoamyl alcohol (25:24:ane, v/v) was added, and the sample was immediately mixed and centrifuged at 5000 rpm in a table-top microcentrifuge for x min. To the upper (aqueous) phase 300 μL of chloroform/isoamyl booze (24:1) was added, which was mixed and centrifuged at 5000 rpm. To the resultant aqueous phase 300 μL of isoamyl booze was added; after mixing, the sample was incubated at room temperature for x min to precipitate DNA. To pellet Deoxyribonucleic acid, the sample was centrifuged at 5000 rpm for 10 min. The pellet was resuspended in 500 μL of 70% ethanol and centrifuged at 5000 rpm for ten min. The supernatant was discarded, and the pellet was stale and dissolved in 20 μL of TE buffer.

Plasmid transformation and isolation

The competent DH5α cells were prepared by calcium chloride method [24]. The ligation product (10 μL) was added directly to 50 μL of competent DH5α cells, incubated for fifteen min on ice, heat-shocked at 42 °C for 1 min, and then transferred on water ice for 5 min. Subsequently calculation 500 μL of LB, the cells were incubated in a shaker at 37 °C for 60 min. Later on incubation, cells were pelleted and resuspended in 100 μL of LB, which was and so spread on LB plates containing ampicillin (100 μg/mL). After overnight incubation at 37 °C, eight colonies from each transformation were randomly picked and checked past colony PCR. Plasmids were isolated using the Spin Miniprep kit (Qiagen).

Abbreviations

bp:: Base pair
CTAB:: Hexadecyltrimethylammonium bromide
dNTP:: Mix of two'-deoxynucleoside 5'-triphosphates
kb:: Kilobase
LB:: Lysogeny broth.
MRF:: Modified restriction-free cloning
PCR:: Polymerase chain reaction
RF:: Brake-free cloning

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Acknowledgments

The authors wish to thank Esteve Padros (Department of Biochemistry and Molecular Biology, Medical School, and Center of Biophysics, Democratic University of Barcelona) for critical reading of the manuscript.

Funding

This piece of work was supported by Starting Grant from Hebei Agricultural Academy to Fanli Zeng (grant number ZD201622) and grants from the National Natural Science Foundation of Communist china to Jingao Dong (grant number 31271997) and Zhimin Hao (grant number 31301616).

Availability of data and materials

The data sets supporting the results of this article are included within the article and its boosted files.

Authors' contributions

FZ, JD, and YL designed the experiments and drafted the manuscript. FZ, ZH and PL carried out the practical work. YM was involved in the research discussions and helped to finalize the manuscript. All authors read and approved the manuscript.

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The authors declare that they take no competing interests.

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Authors and Affiliations

College of Life Sciences, Hebei Agronomical University, Baoding, 071001, Prc

Fanli Zeng, Zhimin Hao, Pan Li, Yanan Meng & Jingao Dong
the Academy of Texas Houston McGovern Medical Schoolhouse, Houston, 77030, Usa

Yibin Lin

Corresponding authors

Correspondence to Jingao Dong or Yibin Lin.

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Zeng, F., Hao, Z., Li, P. et al. A restriction-free method for factor reconstitution using two unmarried-primer PCRs in parallel to generate compatible cohesive ends. BMC Biotechnol 17, 32 (2017). https://doi.org/10.1186/s12896-017-0346-5

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Received: 08 December 2016
Accepted: 01 March 2017
Published: 17 March 2017
DOI : https://doi.org/10.1186/s12896-017-0346-five

Keywords

Restriction-free cloning
Gene reconstitution
Single-primer PCR
Loftier-throughput cloning