AFEAP cloning: a precise and efficient method for large DNA sequence assembly
© The Author(s). 2017
Received: 9 August 2017
Accepted: 30 October 2017
Published: 14 November 2017
Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts.
The AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids.
We characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%.
AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.
DNA sequence assembly, which refers to the precise aligning and merging multiple fragments of DNA, in an end-to-end fashion, into large synthetic circuits and pathways, plays a pivotal role in protein structure-function, metabolic engineering, and synthetic biology [1–5]. The increasingly high demand for assembling large DNA into functional devices requires the methods that allow scarless, sequence independent, multi-fragment assembly of large constructs at high efficiency and high fidelity [6, 7]. In the past decade, many novel DNA assembly methods, such as: Gibson Assembly (GA) , Golden Gate assembly , uracil-specific excision reagent cloning (USER) , ligase cycling reaction (LCR) , DNA assembler , twin-primer assembly (TPA) , sequence and ligation-independent cloning (SLIC) , seamless ligation cloning extract (SliCE) , enzyme-free cloning (EFC) , polymerase incomplete primer extension (PIPE) , in Vivo assembly (IVA cloning) , DNA assembly with thermostable exonuclease and ligase (DATEL) , and overlap extension PCR and recombination (OEPR Cloning) , have been designed and developed (Additional file 1: Table S1), which opened doors to a wide variety of applications. These methods differ in both mechanism and scale, providing the effective means to cope with different needs [2, 4]. Recent advances in synthetic biology would be aided by these new techniques . Despite the advantage of these assembly techniques, to our knowledge, no one approach can satisfy all even most of the requirements and each still has its limitations. As such, the development of novel, easy-to-use, scarless assembly methods with high efficiency and accuracy, especially for multiple fragments and large DNA, are always required .
In the present study, inspired by the concept of restriction-free cloning method  and recent advances in high fidelity DNA polymerase, such as G-HiFi™ DNA polymerase (up to 40 kb DNA with fidelity), we have designed and developed a novel, simple and robust protocol for the construction of large biochemical pathways, circuits, and plasmids. This system requires two rounds of PCRs to generate DNA fragments with compatible 5′ cohesive ends for scarless assembly of multiple DNA fragments with large size into a transformable plasmid. Since the system requires two rounds of PCRs followed by ligation of the sticky ends of DNA fragments, we named the method AFEAP cloning (Assembly of Fragment Ends After PCR). With this “hand in hand” cloning, constructions of an 8 kb plasmid from 1 to 12 fragments, four plasmids with varying sizes of 11.6, 19.6, 28, and 35.6 kb, and a 200 kb of bacterial artificial chromosome (BAC) were achieved with high fidelity.
Results and discussions
Overview of AFEAP cloning method
Determination of parameters for effective assembly
We first tested the effects of the overhang length. The tested DNA overhangs length ranges from 0 to 20 bp. The overhang length was showed marked effects on assembly efficiency (Fig. 2b and c). An overhang, which is less than 2 nucleotides in the PCR products is insufficient for assembly, thereby resulting in low positive clones. From 4 nucleotides overhang onwards, a sharp increase of the efficiency of AFEAP cloning is observed up to 10 bp, with the efficiency peak at 9000 CFUs and 98% of colonies correct. From 10 nucleotides overhang onwards, longer overhangs used somehow decrease the efficiency slightly. As a result, 5–8 nucleotides overhang is, therefore, suitable for AFEAP cloning with high efficiency and low cost. And then we investigated the effects of the size of DNA fragments. Five different size points, i.e., 5.5, 8.0, 15, 20, and 30 kb were tested. The CFU did decrease significantly with longer DNA size fragments (Fig. 2b), while the fidelity did not change significantly within the length of overhang range tested (Fig. 2c). Moreover, we evaluated the effects of the overhangs designed as 5′ end of G/C or A/T. The assembly efficiency of DNA fragments, specifically for those of longer DNA size fragments, is benefiting from 5′ end of the overhang as a G or C (Fig. 2d). In addition, we tested the effects of the ligase treatment. As shown in Fig. 2e, the assembly efficiency for different size fragments did increase significantly when treated with ligase. Last, we evaluated the effects of transformation conditions, such as electroporation or chemical transformation, on the assemble efficiency. Electroporation gave higher efficiencies, but lower fidelities (Fig. 2f).
Summary of optimal conditions for DNA assembly with AFEAP cloning
ranges from 0 to 20 nucleotides
Size of DNA fragments
5.5, 8.0, 15, 20, and 30 kb
Decreased with the increase of DNA size
Overhangs designed as 5′ end of G/C or A/T
overhangs designed as 5′ end of G/C or A/T
T4 DNA ligase treated or not
Ligation ratio (Shorter to longer)
1:6; 1:3; 1:1; 3:1; 6:1; 10:1, 15:1; and 20:1
DNA fragment number
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12
Decreased with the increase of fragments number
Assembly of multiple fragments
After developing and optimizing the AFEAP cloning method, its efficiency and accuracy in assembling multiple fragments were evaluated. We tested the effects of DNA fragment number, final plasmid size, the molar ratio between longer and shorter DNA fragments, and transformation conditions.
Construction of larger plasmid
We have developed an alternative DNA assembly method, named AFEAP cloning, which relies on two-round PCRs to insert complementary sticky ends to each 5′ end of DNA fragments for assembling multiple DNA fragments into functional parts. AFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.
Bacterial strains, plasmids, and reagents
Host strain E. coli DH5α was obtained from Invitrogen (Carlsbad, CA, USA). The competent DH5α cells were prepared by using calcium chloride method . Plasmid pET22b was purchased from Millipore Sigma (Billerica, MA, USA), and pCC1BAC™ vector was purchased from Epicentre® (Madison, WI, USA). Genome DNAs were purchased from American Type Culture Collection (ATCC®, Manassas, VA, USA). Bacteria containing plasmids were cultured in Lysogeny Broth (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) medium with appropriate antibiotics (kanamycin or ampicillin at 50 or 100 μg/ml) when necessary. Phusion® high-fidelity DNA polymerase, DNA marker and T4 DNA ligase were purchased from New England Biolabs (NEB, Ipswich, MA, USA). G-HiFi™ DNA polymerase was purchased from SMOBIO Technology (Hsinchu, Taiwan). QIAquick PCR purification kit, QIAquick gel extraction kit, and QIAprep spin miniprep kit were purchased from Qiagen (Hilden, Germany). Gibson assembly master mix was from NEB (Ipswich, MA, USA).
Primer design and DNA manipulation
All the primers used in this study were designed as shown in Fig. 1b, listed in Supplementary Additional file 2: Table S2, and synthesized by Shanghai Sangon Biotechnology. To determine the optimal length of overhang sequence for AFEAP cloning, the primers designed for first-round PCR flanking overhang region, and the primers designed for second-round PCR carry additional 0 to 20 nucleotides in their 5′ extension (See Fig. 2a for a schematic diagram of primer design). For multiple-fragment assembly, the primers designed for first-round PCR flank overhang regions, and for second-round PCR carry additional 5–8 nucleotides with 5′ end as G or C.
Unless otherwise stated, 50 μL PCR reactions were performed using Phusion® high-fidelity DNA polymerase (NEB). The PCR conditions were listed in Additional files 7 and 8: Tables S3 and S4. The products of first-round PCR were purified by 1% agarose gel extraction with QIAquick gel extraction kit. The complementary DNA products from second-round PCRs were annealed without purification using the condition listed in Additional file 9: Table S5. The DNA fragments with complementary sticky ends were assembled via ligase cycling reaction, which was performed in a final volume of 20 μL using T4 DNA ligase following the standard protocol from New England Biolabs. In brief, the longer and shorter DNA fragments were mixed at a molar ratio of 1:10. The mixture was incubated at room temperature for 2 h. After heat inactivation at 65 °C for 10 min, the reaction was chilled on ice.
Plasmid transformation, isolation, and sequence
After ligation, 10 μL of the ligation products was directly added to 100 μL of competent DH5α cells, incubated for 15 min on ice, heat-shocked at 42 °C for 1 min and then transferred to ice for 5 min. After adding 500 μL of LB medium the cells were subsequently incubated at 37 °C and 200 rpm for 1 h. After incubation, cells were pelleted. The supernatant was removed leaving 100 μl and the pellet was resuspended in the remaining supernatant that was then spread onto a LB agar plate containing ampicillin (100 μg/ml) or kanamycin (50 μg/ml). After incubating the plates overnight at 37 °C, for each transformation we selected ten colonies at random and the plasmids were isolated with QIAprep spin miniprep kit. For each assembly the re-joining junction sites were validated by DNA sequence to ensure accuracy of corrected assembly.
Gibson assembly was carried out according to the manufacturer’s protocol (Gibson Assembly Master Mix Instruction Manual, NEB). In brief, the longer and shorter DNA fragments were mixed at a molar ratio of 1:3–1:10. DNA assembly was performed in a total volume of 20 μL containing 1 pmol DNA fragments and 10 μL Gibson assembly master mix. Samples were incubated in a thermocycler at 50 °C for 60 min. Following incubation, samples were stored on ice or at −20 °C for subsequent transformation.
Electroporation was carried out followed a protocol from New England Biolabs (NEB, Ipswich, MA, USA). In brief, the ligation mixture was purified with QIAquick PCR purification kit, and transformed into electrocompetent DH5α cells by using a Gene Pulsar apparatus (Bio-Rad).
We thank Professor Huimin Zhao (University of Illinois at Urbana − Champaign, USA), Dr. Leslie A. Mitchell (New York University Langone Medical Center, USA) and Dr. Zhen Kang (Jiangnan University, China) for their meaningful discussion.
This work was supported by National Modern Agriculture Industry Technique Systems (CARS-02) to Jingao Dong and Starting Grant from Hebei Agricultural University to Fanli Zeng (grant number ZD201622).
Availability of data and materials
The data sets supporting the results of this article are included within the article and its additional files.
FZ, JD, and YL designed the experiments and drafted the manuscript. FZ, JZ, SZ and HZ carried out the practical work. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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