a simple and efficient method for in vitro site-directed ...site-directed mutagenesis, which...
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A simple and efficient method for in vitro site-directed mutagenesis
Dave Palis, and Frank Huang*
*Correspondence: Frank Huang; [email protected]
Address: Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular,
Facultat de Medicina, and Centre d’Estudis en Biofísica, Universitat Autònoma de
Barcelona, 08193 Bellaterra, Barcelona, Spain.
Abstract
Site-directed mutagenesis, which provides a means of introducing specific nucleotide
changes into a gene, has proven to be valuable for analyzing the changes in function,
stability and/or activity of the target protein. The QuikChangeTM method and its later
modifications are popular used for site-directed mutagenesis, but imperfect. We have
developed an alternative cloning method to perform site-directed mutagenesis based on a
two PCR-round procedure, followed by ligation of the DNA fragments. The first PCR
yields linear DNA fragments with the desired mutations, and is followed by a second
asymmetric (one primer) PCR that inserts overlapping overhangs at both sides of each
DNA fragment. The result of the second PCR is then annealed and ligated with T4 DNA
ligase, followed by bacterial transformation to yield the desired plasmids. We
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demonstrated its application to site-directed mutagenesis, substitutions, insertions and
deletions, for both single and multiple modifications. A significant number of examples
are shown for each of the procedures. Using this method, we show great success in
complicated scenarios such as mutagenesis in larger plasmids, multi-fragment assembly,
and multi-site mutagenesis of up to six simultaneous mutations. The average cloning
efficiency was higher than 95%, as confirmed by DNA sequencing of the inserts. LFEAP
mutagenesis is a complete system, and offers significant advantages for basic
molecular cloning and mutagenesis procedures. LFEAP mutagenesis also provides an
efficient cloning method for complicated scenarios. This method is simple, stable,
efficient and also seamless, and requires no special kits, enzymes or proprietary
bacteria, which makes this method suitable for high-throughput cloning and structural
genomics.
Keywords: LFEAP mutagenesis; single-primer PCR; site-directed mutagenesis; multi-
site mutagenesis; multi-fragment assembly
Background
Polymerase chain reaction (PCR)-based site-directed mutagenesis is an essential
technique in modern genetics and protein engineering. It is widely used to modify the
DNAs sequence, and hence the structure and activity of individual proteins in a
systematic way, opening up opportunities for investigating the structure-function
relationships, enzyme substrate selectivity, or for protein engineering[1-4].
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A number of strategies and commercial kits have been developed to generate site-
directed point mutations, short additions or deletions with the QuikChangeTM site-
directed mutagenesis system developed by Stratagene (La Jolla, CA) which probably is
the most favored method[5]. It uses a high-fidelity DNA polymerase, such as KOD hot
start DNA polymerase, Pfu DNA polymerase, or Phusion® high-fidelity DNA
polymerase, etc., to amplify the whole plasmid by regular PCR using a pair of full
complement primers with the desired mutations in the center (Agilent Technologies).
The resulting DNA pool (parental plasmids and mutant strands with nick) is then
treated with DpnI to digest the methylated parental templates and transformed into
competent cells, where the host cells repair the nick to yield the plasmid with the
desired mutation.
QuikChangeTM system has some limitations, despite its wide use[5, 6]. Since the
primers completely overlap, and hence favor self-annealing, this method limits the
yield of amplified product and gives rise to false positives[6, 7]. The use of
complementary primer pairs may lead to the formation of "primer dimers" by partial
annealing of a primer with the second primer in reaction, and formation of tandem
primers repeats, reducing the efficiency of successful transformants[8]. As the newly
synthesized DNA is "nicked", it cannot be used as a template for subsequent
amplification in contrast to "normal" PCR, leading to a less efficient PCR
amplification[5]. In practice, the generation of desired mutation frequently fails when
PCR amplification efficiency is low[9, 10]. In addition, the originally developed
QuikChange™ cannot introduce multiple mutations[5, 6, 11].
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To circumvent these limitations, many modified versions of QuikChangeTM site-
directed mutagenesis method have been developed[5, 12-14]. These methods use partially
overlapping primers to reduce the formation of primer dimers, and improve PCR
amplification efficiency. Recently, several labs have reported new alternative methods
for generating site-directed mutagenesis. Overlap extension PCR was reported as an
effective method for generating single and multiple-site plasmid mutagenesis[15-17].
Recombineering systems in vivo and in vitro were reported as powerful tools for
generating site-directed modifications in plasmids in a cost-efficient manner with high
accuracy[7, 18-20]. These methods circumvent the limitations associated with
QuikChangeTM site-directed mutagenesis method to some extent, and have made site-
directed mutagenesis more accessible.
Recently, we described a restriction-free cloning method for gene reconstitution[6].
This approach can be adopted to insert any DNA fragment up to 20 kb into a plasmid in
the absence of unwanted alterations to the vector backbone. In this study, we used this
method to develop a complete system for site-directed mutagenesis. This system requires
two rounds of PCRs to generate mutated DNA fragments with compatible 5' or 3'
cohesive ends, for scarless assembly of multiple modified DNA fragments 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 LFEAP mutagenesis
(Ligation of Fragment Ends After PCR). By using this method, we were able to generate
a variety of DNA modifications (point mutations, substitutions, deletions, and insertions)
in vectors in a cost-efficient manner with high accuracy.
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Results
Method overview and primer design. To implement LFEAP mutagenesis, we first
optimized primer design and investigated the efficiency of our scarless DNA
modification method to assemble multi-part DNA. We provide examples from our
work to modify large gene clusters from the E.coli genome, as well as single genes
(yaaU, ileS, talB and apaG from E.coli genome and GAST, MCM6, PRRT2, and
SLC18A2 from human cDNA) , in a variety of expression vectors for different types of
mutations.
LFEAP mutagenesis uses in vitro assembly of PCR amplified DNA fragments,
guided by short complementary flanking regions that are fused together by ligation. All
types of DNA modifications proceed similarly through two rounds of PCRs: regular
double-primer PCR for target DNA fragments amplification is followed by two single-
primer linear PCRs in parallel to generate overhang cohesive ends for ligation (Fig. 1).
The crucial point for successful LFEAP mutagenesis is to define an “overhang” region.
As shown in Fig. 1, the overhang region can be a short sequence of 5-8 nucleotides on the
3' terminus of the mutated site (like single point mutation, deletion, as well as substitution
and insertion of short sequence; Fig. 1A and B), inside a DNA fragment (like insertion
and substitution of long sequence; Fig. 1C), or right on the cloning sites (like subcloning;
Fig. 1D).
As outlined in Fig. 1, all DNA modifications and overhang regions are introduced at
the 5′ end of the primers. The forward (Fw1) and reverse (Rv1) primers used for first-
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round PCR are designed following common guidelines employed in double-primer PCR
with DNA modifications at the 5′ end[8]. Primers (Fw2 and Rv2) designed for the
second-round PCRs in parallel are essential for successful mutations. For site-directed
point mutation, substitutions of short sequences, and insertions of short sequences, both
primers include overhang region and the Fw primer contains desired codon sequence in
replace of the original one between the template annealing oligo and overhang region
(Fig. 1A). Similarly, for deletions, both primers include overhang region at the 5′ end and
Fw primer escapes the region to be deleted (Fig. 1B). For substitutions and insertions of
longer sequences, the 5-8 nucleotides overhang is picked from the center of the sequence
to be replaced. In this case, the template annealing oligo of both primers are flanked with
overhang region and the desired insertion or substitution sequences from one side of
overhang region (Fig.1C). The maximum size of the insertion is largely dictated by
oligonucleotide synthesis limitations. For subcloning, two pairs of primers are used to
amplify vector and insert individually (Fig.1D). In all, the primers used for second-round
PCRs are designed nearly the same as those for the first-round PCRs, but only with the
addition of overhang sequence at their 5′ ends.
After two-round PCRs and annealing following the scheme of Fig. 1, the newly
synthesized PCR products with sticky ends therefore enable multi-part DNAs to assemble
into a transformable plasmid in vitro. These new reconstituted plasmids were then
transformed into competent E. coli cells and the presence of modification can be
confirmed by DNA sequencing.
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Optimal overhang sequence for LFEAP mutagenesis. To determine the optimal
overhang sequence for higher efficiency, we tested 8 primer pairs with overhang ranging
from 2 to 20 nucleotides (See Supplementary Table S1 for primer sequences). The
percentage of clones containing the desired mutations over total sequenced ones was
calculated as the efficiency with respect to oligo number of overhang incorporated in the
PCR products (Fig. 2). A 2-nucleotide overhang in the PCR products is insufficient for
recombination, thereby resulting in low positive clones. From 4 nucleotides overhang
onwards, a sharp increase of the efficiency of LFEAP mutagenesis was observed up to
10 bp, with the efficiency peak at 98%. Based on these data, 5-8 nucleotides overhang is
therefore suitable for LFEAP mutagenesis with high efficiency. Longer overhangs used
somehow decrease the efficiency slightly (Fig. 2).
Subcloning. Before applying this new approach for site-directed mutagenesis, we
confirmed the ability of LFEAP mutagenesis to subclone our target genes, i.e. yaaU,
ileS, talB, apaG, GAST, MCM6, PPRT2, and SLC18A2 into target vectors. We used
LFEAP mutagenesis to insert E.coli gene of yaaU, ileS, talB, or apaG into
pBAD/Myc-His between 321G and 424T, and human gene of GAST, MCM6, PPRT2,
or SLC18A2 into pcDNA™ 3.1 (+) between 901G and 952G (Supplementary Table
S4). The primers designed in accordance with the strategy of LFEAP mutagenesis and
PCR conditions used for subcloning are listed in Supplementary Table S1 and Table
S2, respectively. The DNA products of two-round PCRs and ligation were evaluated
by 1% agarose gel electrophoresis and cloning sites were verified by sequencing (Fig.
3A and Supplementary Figure S1). The assemblies between inserts and vectors with
sticky ends obtained from two-round PCRs, lead to band-shift (see Fig. 3A and
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Supplementary Figure S1). We obtained hundreds of colonies in one transformation
and average 95% positive colonies (of 10 colonies tested) as verified by sequencing for
all examples (Supplementary Table S4).
Site-directed point mutations. Site-directed point mutations are widely used to
characterize protein-protein interactions, protein functional sites, or active sites of
enzymes over the last three decades[21-23]. In the present work, we evaluated the
suitability of LFEAP mutagenesis for generating site-directed point mutations.
Here we show examples of point mutations in seven protein-coding genes from
different species: yaaU (R205A), ileS (K581A), talB (K193C), and apaG (R26A) from
prokaryotic E.coli and GAST (K75A), MCM6 (Q641A), and SLC18A2 (K354A) from
eukaryotic human cells (using the vectors pNGFP-BC-yaaU, pNGFP-BC-ileS, pCGFP-
BC-talB, pCGFP-BC-apaG, pNGFP-EU-GAST, pNGFP-EU-MCM6, pCGFP-EU-
SLC18A2) (Supplementary Table S5). The primers and PCR conditions used for
creating point mutations are listed in Supplementary Table S1 and Table S2,
respectively. Products from two-round PCRs were separated in the gels shown in Fig.
3B and Supplementary Figure S2. These linear DNA fragments with sticky ends were
circularized by T4 DNA ligase, which leads to band-shift on the agarose gel (see Fig.
3B and Supplementary Figure S2). The presences of desired mutations were verified
via DNA sequencing (see Fig. 3B and Supplementary Figure S2). 187-389 CFUs
(colony formation units) were obtained within one transformation, of which 98.5% (of
10 colonies tested) on average contained the correct sequence (see Supplementary
Table S5).
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We compared commercial QuikChangeTM mutagenesis method with LFEAP
mutagenesis for the same mutants. The efficiency of QuikChangeTM mutagenesis
method was shown in Supplementary Table S6. LFEAP mutagenesis shows higher
efficiency under our test conditions compared to QuikChangeTM mutagenesis. Fewer
colony numbers (2-21) and lower accuracy of 32.6% were obtained for all
QuikChangeTM tests (Supplementary Table S6).
Substitutions. Substitutions are common modifications, used for modifying gene
promoters, protein structures, or domains[24, 25]. We evaluated the efficiency of
LFEAP mutagenesis for substitution. All substitutions were targeted to plasmids of
pNGFP-BC-yaaU and pNGFP-EU-GAST encoding E.coli yaaU protein and
human GAST protein (Supplementary Table S7). The primers and PCR conditions
used for creating point mutations are listed in Supplementary Table S1 and
Supplementary Table S2, respectively.
First, we substituted six nucleotides in yaaU (670GATGAA with GCCGCA) and
nine nucleotides in GAST (79TCCCAGCAG with GCCGCAGCG), which results in
mutants of yaaU DE224AA and GAST SQQ27AAA. The DNA products obtained
from each step of LFEAP mutagenesis were separated in the gels as shown in Fig. 3C
and Supplementary Figure S3. These substitution mutations were verified by DNA
sequencing (Fig. 3C and Supplementary Figure S3). We obtained 100% for yaaU
DE224AA and 90% for GAST SQQ27AAA correct mutagenesis (of 10 colonies
tested) (Supplementary Table S7). As substitution of a few nucleotides by LFEAP
mutagenesis worked as efficiently as point mutation, we proceeded to generate larger
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editing of the DNA sequence using the same approach. We substituted thirty
nucleotides in the yaaU gene resulting in YAAU
RKGRVKECEE202AAAAAAAAAA mutant and thirty-six nucleotides in the GAST
resulting in EQQGPASHHRRQ48AAAAAAAAAAAA mutant. Under our test
conditions, we got hundreds of colonies and almost all of them were positive as
verified by sequencing (Supplementary Table S7).
Deletions. LEFAP mutagenesis is not limited to substitution. We performed deletion
mutations taking advantage of this method. All deletions were targeted to plasmids of
pNGFP-BC-yaaU, pNGFP-BC-ileS, pCGFP-BC-talB, pCGFP-BC-apaG, pNGFP-EU-
GAST, pNGFP-EU-MCM6, pNGFP-EU-PPRT2, pCGFP-EU-SLC18A2
(Supplementary Table 8). The primers and PCR conditions used for creating deletions
are listed in Supplementary Table S1 and Supplementary Table S2, respectively. The
PCR products were evaluated by 1% agarose gel electrophoresis and mutations were
verified by sequencing (Fig. 3D, Supplementary Figure S4).
First, single nucleotide in the yaaU 909A, ileS 2096T, talB 552C, apaG 253G,
GAST 183A, MCM6 1745T, PPRT2 741C, and SLC18A2 1415G was deleted, which
results in a frame-shift of the C-terminal tail of the encoded proteins. Significant
colony numbers (117-247) and average 93.8% correct mutagenesis (of 10 colonies
tested) were obtained (Supplementary Table S8). LEFAP mutagenesis is therefore an
efficient method for generating deletion of single nucleotide. We then proceeded
longer sequence deletion.
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12 nucleotides deletion of yaaU gene, ileS gene, talB gene, apaG gene, GAST
gene, MCM6 gene, PPRT2 gene, and SLC18A2 gene, resulting in yaaU (Del F28-G31),
ileS (Del R202-R205), talB (Del Q28-D31), apaG (Del G63-G66), GAST (Del H55-
R58), MCM6 (Del D202-K205), PPRT2 (Del D43-E45), and SLC18A2 (Del D73-
Q76) were achieved by using LEFAP mutagenesis. For each mutant, we obtained
hundreds of colonies where most were correct as verified by sequencing
(Supplementary Table S8).
Finally, we used LFEAP mutagenesis to delete 1272 nucleotides from pNGFP-
BC-yaaU, 2748 nucleotides from pNGFP-BC-ileS, 885 nucleotides from pCGFP-BC-
talB, 309 nucleotides from pCGFP-BC-apaG, 238 nucleotides from pNGFP-EU-
GAST, 2397 nucleotides from pNGFP-EU-MCM6, 954 nucleotides from pNGFP-EU-
PPRT2, 1476 nucleotides from pCGFP-EU-SLC18A2, resulting in yaaU (Del K11-
N434), ileS (Del G14-A929), talB (Del V14-K308), apaG (Del V14-F116), GAST
(Del G14-L92), MCM6 (Del Q14-V812), PPRT2 (Del V14-S331), and SLC18A2 (Del
E14-I505). We obtained hundreds of colonies in one transformation and average
98.5% correct mutagenesis (of 10 colonies tested) as verified by sequencing for tested
examples (Supplementary Table S8).
Insertions. We also tested whether LFEAP mutagenesis can be used to generate
insertion mutants. Insertion cases as examples were targeted to plasmids of pNGFP-
BC-yaaU, pNGFP-BC-ileS, pCGFP-BC-talB, pCGFP-BC-apaG, pNGFP-EU-GAST,
pNGFP-EU-MCM6, pNGFP-EU-PPRT2 and pCGFP-EU-SLC18A2 (Supplementary
Table 9). The primers and PCR conditions used for creating insertions are listed in
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Supplementary Table S1 and Table S2, respectively. The DNA products of two-round
PCRs and ligation were evaluated by 1% agarose gel electrophoresis and mutations
were verified by sequencing (Fig. 3E, Supplementary Figure S5).
First, we inserted a single nucleotide in yaaU (909A), ileS (2096T), talB (552C),
apaG (253G), GAST (183A), MCM6 (1745T), PPRT2 (741C), and SLC18A2 (1415G),
which results in a frame-shift of the C-terminal tail. These insertions were efficiently
achieved by using LFEAP mutagenesis. Hundreds of colonies (129-218) and average
96.3% correct mutagenesis (of 10 colonies tested) were obtained (Supplementary
Table S9). We then performed larger editing of the target DNA sequences following
the same procedure.
We inserted 12 nucleotides (GCCGCAGCGGCC) into yaaU gene, ileS gene,
talB gene, apaG gene, GAST gene, MCM6 gene, PPRT2 gene, and SLC18A2 gene,
resulting in yaaU (Ins F28-AAAA), ileS (Ins E201-AAAA), talB (Ins Q28-AAAA),
apaG (Ins Q63-AAAA), GAST (Ins H55-AAAA), MCM6 (Ins D202-AAAA), PPRT2
(Ins D43-AAAA), and SLC18A2 (Ins D73-AAAA). For each mutant, we obtained
hundreds of colonies where most of them were correct as verified by sequencing
(Supplementary Table S9).
Finally, we used LFEAP mutagenesis to insert 60 nucleotides
(GTTGAGGAGAGTCCCAAGGTTCCAGGCGAAGGGCCTGGCCATTCTGAAGC
TGAAACTGGC) into yaaU gene and SLC18A2 gene, which results in mutant of yaaU
(Ins F28-VEESPKVPGEGPGHSEAETG) and SLC18A2 (Ins D73-
VEESPKVPGEGPGHSEAETG). Significant large colony numbers and high accuracy
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of average 95% (of 10 colonies tested) were obtained for these two insertion mutations
(Supplementary Table S9).
Multiple-site modifications. Multiple-site modifications on a plasmid are frequently
required with applications in the studies of protein-nucleic acid interactions, protein
structure-function relationships, or protein-protein interactions[26-28]. Current
common strategies for generating multiple-site modifications are based on overlap
extension PCR[16, 17], which requires multiple rounds of PCRs to fuse each DNA
fragments and further gel purification of DNA products from each round PCR. Our
presented work show that LFEAP mutagenesis is simple and efficient for almost all
types of site-directed mutagenesis (site-directed point mutation, insertion, and
deletion) as well as gene subcloning merely by two-round PCRs and ligation. To test
the feasibility of LFEAP mutagenesis for multiple-site modifications on a plasmid, we
proceeded to generate six points mutations in a vector (see Fig. 4A for a schematic
detailing of the cloning procedure) and assembled two genes into a vector (see Fig. 5A
for a schematic details of the cloning procedure) by using our presented strategy.
Here we show example of six point mutations in human MCM6 gene (Q70A,
Q209A, Q342A, D463A, Q597A, and R732A) generated on pNGFP-EU-MCM6. The
primers designed in accordance with the strategy of LFEAP mutagenesis and PCR
conditions used for creating substitutions are listed in Supplementary Tables S1 and
Tables S2. DNA products of two-round PCRs and following assemblies were analyzed
by 1% agarose gel shown in Fig. 4B. Overhangs guide assembly of five DNA
fragments to form the desired propagative construct, which leads to band-shift (Fig.
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4B, lane 15). Presence of cloning sites were verified via DNA sequencing (Fig. 4C).
103 CFUs were obtain on transformation with 100% positive containing deserved
modifications (10/10 colonies tested; Supplementary Table S10).
More complex modifications can be generated by using LFEAP mutagenesis.
We built a pET22b-FLAG-T4L-GGSGGlinker-MCM6 tandem construct, encoding T4
lysozyme (T4L)[29] and MCM6 protein fused by a peptide linker, from the pET22b-
T4L and the pNGFP-EU-MCM6 plasmids. For this we designed primers 1 to remove
the N-terminal domain coding region of T4L and replace it with a FLAG-tag[30]
(DYKDDDDK, tag encoded sequence in primers as insertion), and primers 2 to
subclone the MCM6 open reading frame after the T4L coding region, with a GGSGG
linker at the fusion site (linker encoded in primers as per insertions) (see Fig. 5A for a
schematic detailing of the cloning procedure). The primers and PCR conditions used for
creating substitution are listed in Supplementary Tables S1 and Table S2. DNA
products of two-round PCRs and following assemblies are analyzed by 1% agarose gel
shown in Fig 5B. After two-round PCRs, overhangs guide assembly of three DNA
fragments to form the desired propagative construct (Fig. 5B, lane 8). Presence of
cloning sites were verified via DNA sequencing (Fig. 5C). 57 CFUs were obtained
within one transformation, of which 90% (9/10 colonies) contained the correct
sequence (see Supplementary Table S10).
Modification of larger plasmid. To test the feasibility of LFEAP mutagenesis for
larger plasmid modification, a pET22b vector, between the start codon ATG (287), and
the sequence (157) CACCACCACCACCACCAC, inserted with a 20 kb DNA fragment
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of E. coli genome (200485-220925) containing 21 genes (Supplementary Table S11),
was used to introduce a mutation, ldcC Q32A within the gene cluster.
The two nucleotides (GC) were introduced to replace CA present in the original
vector (AGGCTTTCAGATTATCTGG) such that the resulting sequence
(AGGCTTTGCGATTATCTGG) leaves a mutant of ldcC Q32A in the plasmid. The
primers and PCR conditions used for creating substitution are listed in Supplementary
Tables S1 and Table S2. First-round and second-round PCRs result in single band
(25.5 kb; lane 1 and lane 2 in Fig. 6A), respectively. The linear DNA with sticky ends
was cyclized by T4 DNA ligase and shifted to a higher molecular weight (Lane 4 in
Fig. 6A). The presence of ldcC Q32A was confirmed by DNA sequencing (Fig. 6B). 67
CFUs and 90% correct mutagenesis (of 10 colonies tested) were obtained within one
transformation. Overall, LEFAP mutagenesis is also efficient to introduce mutations
into large plasmids of up to 25 kb in our conditions.
Discussion
We recently presented a restriction-free cloning method for DNA assembly. This
approach enables annealing of in parallel linear PCR products to generate sticky ends
for guiding DNA fragments assembly, which provides an alternative cloning method
inserting any DNA fragment of up to at least 20 kb into a plasmid, with high
efficiency[6]. Here, we apply this approach to the generation of site-directed
mutagenesis. Since the system requires two-round PCRs followed by ligating of the
sticky ends of DNA fragments, we named the method LFEAP mutagenesis (Ligating
of fragment ends after PCR).
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By using LFEAP mutagenesis, we successfully generated a variety of site-direct
mutagenesis, including point mutations, substitutions, deletions, and insertions,
ranging from one nucleotide modification to long DNA sequence modification within
simple steps. We were also able to add nucleotides up to at least 60 nucleotides. The
limitation of longer nucleotides addition is just on the cost of longer primers
themselves, as all the modifications were incorporated through linear PCR by the
modified primers in our method. LFEAP mutagenesis was used as scarless method for
subcloning[6]. In our present work, this method is suitable for the multi-fragment
assembly, which theoretically can be extended for library construction. LEFAP
mutagenesis is also efficient to introduce mutations into large plasmids of up to 25 kb
in conditions. By using this approach, we achieved a high efficiency of over 95% for
generating all the DNA modifications tested. LFEAP mutagenesis therefore can be
applied not only for complex DNA fragments assembly, but for almost all kinds of
site-directed mutagenesis.
LFEAP mutagenesis is a simple and robust method for site-directed mutagenesis
by ligating of fragments ends after PCR. The critical point for this method is to define
an “overhang” region, which is the key for efficient DNA fragments assembly. For
convenience, the overhang region is standardized to be set on the 3' terminus of the
modification sites (like single point mutation, deletion, as well as substitution and
insertion of short sequence), inside a DNA fragment (like insertion and substitution of
long sequence), or right on the cloning sites (like subcloning) (see Fig. 1). In this way, we
standardized the primers design that all modified nucleotides and overhang regions were
introduced at the 3' end of the template annealing regions. This primer design facilitates
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the introduction of long mutation sequences and leaves 5' end sequence completely
complementary to the template, such that primer-template binding is favored over
primer-primer self-complementarity[18].
As shown in Fig. 2, a 5-8 nucleotides of overhang at the ends of PCR products gave
the maximum cloning efficiency of 98%. The cloning efficiency reaches the plateau, and
decreases slightly when longer overhangs are used due to the possibility of secondary
structures formation during PCR[18]. Traditional PCR based mutagenesis methods
typically require a variety of steps and multiple enzymes such as methylation sensitive
DpnI restriction enzyme to digest parental plasmids and kinases for phosphorylation of
3′ ends[18]. Newly developed recombination-based mutagenesis and cloning methods
such as Gibson Assembly® and GeneArt® seamless cloning also need expensive and
special enzymes[18]. Recent IVA cloning which relies on the presence of the
homologous recombination pathway in E.coli, has been shown as a simple and robust
method for DNA assembly[7]. However, this approach may face the possibility of
undesired homologous recombination between host strains and target genes. LFEAP
mutagenesis uses linear PCR to generate overhang cohesive ends for direct ligation,
hence only high fidelity DNA polymerase and T4 ligase are required that are the most
common enzymes in lab. Our two-round PCRs design actually dilutes parental
templates, which reduces the background and improves cloning efficiency. With this
method, no more special enzymes, plasmids, kits, or host strains are required.
Many PCR-based mutagenesis methods use completely or partially overlaped
primer pair, where primers favor self-annealing, limiting the amount of amplified
products and giving rise to false positives[7]. The strategy of primer design of LFEAP
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
18
mutagenesis greatly reduces the complementary region of the primers and allows full
displacement of the modified nucleotides outside the template annealing region (Fig.
1). This eliminates primer-dimer formation and mispriming, which ensures exponential
amplification for high PCR efficiency.
QuikChangeTM site-directed mutagenesis and its variations[5, 12-14, 31] require
bacterial endogenous DNA repair system to repair the nicks in circular PCR
products[32]. However, the efficiency for the nicks repair by bacterial endogenous
repair machinery is low, which therefore leads to inefficiency particularly for
mutagenesis of more than one nucleotide[18]. Newly developed recombination-based
mutagenesis and cloning methods, like SLIC[33], SLICE[34], REPLACR-
mutagenesis[18], and IVA cloning[7], harness the power of homologous
recombination and are performed either in vitro or within the living cells. But it is
difficult to monitor the efficiency of homologous recombination directly, which could
limit the stability of these methods. While as, LFEAP mutagenesis presented here
efficiently assembles the modified DNA fragments in vitro by traditional ligation
reaction that can be accurately manipulated and monitored by agarose gel
electrophoresis directly. In our present work, we stably obtained hundreds of colonies
and high efficiency for each mutation. Thus, LFEAP mutagenesis is a stable, but
efficient cloning method for generating site-directed mutagenesis.
One of the limiting factors in LFEAP mutagenesis is the PCR itself; most high-
fidelity polymerases are recommended for PCR products up to 20–25 kb, though there
have been some improvements of polymerases development. Nevertheless, most
plasmids used are smaller than 10–12 kb and hence the utility of the method is
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19
sufficient for most routine mutagenesis. The other one disadvantage associated with
LFEAP mutagenesis is that it needs two-step PCR, thus needs one more pair of
primers and one more round of PCR. Luckily, primer synthesis is not any more costly,
at least not more expensive compared to enzymes. LFEAP mutagenesis requires two-
round of PCRs that need more time to complete all cloning procedures. However, this
approach doesn’t require any treatments of enzymes, which always need a couple of
hours. Due to high stability and efficiency, we always obtained deserved mutants in
one time experiment that actually saves a lot time and reduces labour.
In conclusion, LFEAP mutagenesis provides an alternative simple method for
generating site-directed mutagenesis and complex DNA fragments assembly with high
efficiency and accuracy.
Materials and Methods
E. coli strains, primers, plasmids, and reagents. Host strain E. coli DH5α was
obtained from Invitrogen Corp. The competent DH5α cells were prepared by using
calcium chloride method[35]. Bacteria containing plasmids were cultured in Lysogeny
Broth (LB) medium with appropriate antibiotics (Kanamycin or Ampicillin at 50 or 100
μg/ml). Primers were designed using OligoCalc[36] and SnapGene® and were purchased
from Invitrogen Corp. (listed in the Supplementary Table S1). All primers were designed
to bind template DNA at 60 °C. pET22b and pcDNA™ 3.1 (+) were obtained from
Invitrogen Corp. The plasmids of pNGFP-BC, pCGFP-BC, pNGFP-EU, and pCGFP-EU
were courtesy of Dr. Eric Gouaux. Phusion® high-fidelity DNA Polymerase, DNA
marker, Taq DNA polymerase, and T4 DNA ligase were purchased from New England
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20
Biolabs, cloning kits from Qiagen. Human cDNAs were purchased from Clontech. The
PCR purification kit and gel extraction kit were purchased from Qiagen. The plasmids
were isolated using a QIAprep Spin Miniprep Kit (Qiagen).
PCR and ligation. Primer sequences specific for the mutation are listed in
Supplementary Table S1. Unless otherwise stated, 50 μL PCR reactions were performed
using Phusion® High-Fidelity DNA polymerase (NEB). The PCR conditions are listed in
Supplementary Table S2. The products of first-round PCR were purified by 1%
agarose gel extraction. The complementary DNA products from second-round PCRs
were annealed without purification. The DNA fragments with complementary sticky
ends were cycled ligation assembly by T4 ligase (NEB). DNA ligation reactions were
performed to fuse DNA fragments 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 1:3–1:10. The reaction was
incubated at room temperature for 2 hours. After heat inactivation at 65 °C for 10 min,
the reaction was chilled on ice.
Plasmid transformation, isolation, and sequencing. 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, the
cells were incubated on a shaker at 37 °C for 60 min. After incubation, cells were pelleted and
resuspended in 100 µL LB, which was then spread on LB plates 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 using Spin
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21
miniprep kit (Qiagen, Germany). Sequencing was performed to ensure accuracy of the mutated
sequences and the cloning sites.
Determining optimal overhang length needed for LEDPAP cloning. Primers were
designed for the addition of two nucleotides (TA) in the middle of EcoRI restriction
site (GAATTC) in pcDNA™3.1 (+)-MCM6 plasmid, thereby disrupting the restriction
site[37]. The overhang length was varied from 2 bp to 20 bp (See Supplementary Table
S1 for primer sequences). PCR products were subjected to LFEAP mutagenesis protocol
as mentioned above (Fig. 1). The resulting bacterial colonies were analyzed by colony
PCR (forward primer: GAAGGCTACTCTGAACGCCCGGACGTC, reverse primer:
CACTAAATCGGAACCCTAAAGGGAGC; see Supplementary Table S3 for PCR
conditions). The expected PCR product for the mutated plasmids should be 1531 bp
and not amenable to EcoR I digestion whereas the pcDNA™3.1 (+)-MCM6
background PCR product should be 1531 bp and following EcoRI digestion to yield
1000 bp and 531 bp products.
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Authors' contributions
DP and FH designed the experiments and drafted the manuscript. All authors read and
approved the final manuscript.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
24
Conflict of interest: The authors declare no competing interests exist.
Figure Legends
Figure 1. Schematic representation of the LFEAP mutagenesis procedures. Primer
design is shown for each type of basic modification: site-directed substitution and
insertion of short sequence (A), deletion (B), insertion and substitution of long sequence
(C), and subcloning (D). For these modifications, a 5–8 nucleotides on the 3' terminus of
the modification sites (like A and B), inside a DNA fragment (like C), or right on the cloning
sites (like D) is defined as “overhang” region (purple and green). The primer pairs
designed for the first-round PCR don't include overhang region, but contain DNA
modifications at 5' end of forward primer. Primers designed for second-round PCR
include overhang region at their 5' end. Fw: forward primer, Rv: reverse primer, OH:
overhang region, Del: deletion sequence, Ins: insertion sequence,
Figure 2. Effect of overhang length on LFEAP mutagenesis efficiency. The effect of
overhang length involving in PCR products is plotted against the achieved efficiencies
with LFEAP mutagenesis. 5-10 nucleotides of overhangs gave the maximum efficiency,
while the cloning efficiency decreases when longer overhangs used.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
25
Figure 3. Basic molecular cloning procedure using LFEAP mutagenesis. Agarose
electrophoresis resulting (left panel) from the amplification using the primers as listed at
Supplementary Table S1 and DNA sequencing confirmations of the mutation sites (right
panel). (A) subcloning. Lane 1: First-round PCR product of pBAD/Myc-His; Lane 2:
First-round PCR product of yaaU; Lane 3: Annealing of second-round PCR product of
pBAD/Myc-His; Lane 4: Annealing of second-round PCR product of yaaU; Lane 5: 1 kb
DNA ladder; Lane 6: Mixture of DNA samples shown in lane 3 and lane 4 at the molar
ratio of 1:3 before ligation; Lane 7: Mixture of DNA samples shown in lane 3 and lane 4
at the molar ratio of 1:3 after ligation; 8: 1 kb DNA ladder. (B) site-directed point
mutations. (C) substitutions. (D) deletions. (E) insertions. (B) – (E): Lane 1: first-round
PCR product using Fw1 and Rv1 primers; Lane 2: Annealing of PCR products with Fw2
or Rv2 primer using DNA shown in lane 1 as template; Lane 3: DNA sample shown in
lane 2 before ligation; Lane 4: DNA sample shown in lane 2 after ligation; Lane 5: 1 kb
DNA ladder. DNA samples were electrophoresed in 1% agarose gel. Red boxes and
arrow show the cloning sites or mutation sites.
Figure 4. Multiple-site modifications using LFEAP mutagenesis. (A) Schematic
details show the flow chart of the generation of multiple-site mutations using LFEAP
mutagenesis. Five parallel regular double-primer PCR reactions were performed to
amplify each DNA fragment followed by single-primer linear PCR reactions in parallel to
generate overhanging cohesive ends for DNA fragments assembly. (B) Agarose
electrophoresis showing the amplification using the primers as listed at Supplementary
Table S1. Lane 1: PCR products from reaction with primers MCM6 Multi-
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26
R732Afw1/MCM6 Multi-Q70Arv1 using plasmid pNGFP-BC-MCM6 as template; Lane
2: PCR products from reaction with primers MCM6 Multi-Q70Afw1/MCM6 Multi-
Q209Arv1 using plasmid pNGFP-BC-MCM6 as template; Lane 3: PCR products from
reaction with primers MCM6 Multi-Q209Afw1/MCM6 Multi-Q342Arv1 using plasmid
pNGFP-BC-MCM6 as template; Lane 4: PCR products from reaction with primers
MCM6 Multi-Q342Afw1/MCM6 Multi-D463Arv1 using plasmid pNGFP-BC-MCM6 as
template; Lane 5: PCR products from reaction with primers MCM6 Multi-
D463Afw1/MCM6 Multi-Q597Arv1 using plasmid pNGFP-BC-MCM6 as template;
Lane 6: PCR product from reaction with primers MCM6 Multi-Q597Afw1/MCM6 Multi-
R732Arv1 using plasmid pNGFP-BC-MCM6 as template; Lane 7: Annealing of PCR
products with primer MCM6 Multi-R732Afw2 and PCR products with primer MCM6
Multi-Q70Arv2 using DNA shown in lane 1 as template; Lane 8: Annealing of PCR
products with primers MCM6 Multi-Q70Afw2 and MCM6 Multi-Q209Arv2 using DNA
shown in lane 2 as template; Lane 9: Annealing of PCR products with primer MCM6
Multi-Q209Afw2 or MCM6 Multi-Q342Arv2 using DNA as in lane 3 as template; Lane
10: Annealing of PCR products with primers MCM6 Multi-Q342Afw2 or MCM6 Multi-
D463Arv2 using DNA shown in lane 4 as template; Lane 11: Annealing of PCR products
with primers MCM6 Multi-D463Afw2 or MCM6 Multi-Q597Arv2 using DNA shown in
lane 5 as template; Lane 12: Annealing of PCR products with primers MCM6 Multi-
Q597Afw2 or MCM6 Multi-R732Arv2 using DNA shown in lane 6 as template; Lane 13:
1 kb DNA ladder; Lane 14: Mixture of DNA from lane 7, 8, 9, 10, 11, and 12 at the
molar ratio of 1:3:3:3:3:3 before ligation; Lane 15: Mixture of DNA from Lane 7, 8, 9, 10,
11, and 12 at the molar ratio of 1:3:3:3:3:3 after ligation; Lane 16: 1 kb DNA ladder.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
27
DNA samples were electrophoresed in 1% agarose gel. (C) DNA sequencing
confirmations of the mutation sites.
Figure 5. Multiple-fragment assembly using LFEAP mutagenesis. (A) Schematic
details show the flow chart of a multi-fragment assembly using LFEAP mutagenesis. The
independent fragments with 3' end overhangs were amplified parallelly with two-round
PCRs and assembled. (B) Agarose electrophoresis showing the amplification using the
primers as listed at Supplementary Table S1. Lane 1: PCR products from reaction with
primers pet22bfw1/pet22brv1 using plasmid pET22b as template; Lane 2: PCR products
from reaction with primers T4L-Flag-fw1/T4L-Flag-rv1 using plasmid pET22b-T4L as
template; Lane 3: PCR products from reaction with primers MCM6-GGSGG-
fw1/MCM6-GGSGG-rv1 using plasmid pNGFP-BC-MCM6 as template; Lane 4:
Annealing of PCR products with primer pet22bfw2 or pet22brv2 using DNA as in lane 1
as template; Lane 5: Annealing of PCR products with primers T4L-Flag-fw2 or T4L-
Flag-rv2 using DNA as in lane 2 as template; Lane 6: Annealing of PCR products with
primers MCM6-GGSGG-fw2 or MCM6-GGSGG-rv2 using DNA shown in lane 3 as
template; Lane 7: Mixture of DNA from Lane 4, 5, and 6 at the molar ratio of 1:3:3:3
before ligation; Lane 8: Mixture of DNA from Lane 4, 5, and 6 at the molar ratio of
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
28
1:3:3:3 after ligation; Lane 9: 1 kb DNA ladder. DNA samples were electrophoresed in 1%
agarose gel. (C) DNA sequencing confirmations of the cloning sites.
Figure 6. Modification of larger plasmids. (A) Agarose electrophoresis shows the
amplification using the primers as listed at Supplementary Table S1. Lane 1: PCR
products from reaction with primers ldcCQ32Afw1/ldcCQ32Arv1 using plasmid
pET22b inserted 20 kb genes cluster as template; Lane 2: Annealing of PCR products
with primer ldcCQ32Afw2 or ldcCQ32Arv2 using DNA shown in Lane 1 as template;
Lane 3: DNA sample from Lane 2 before ligation; Lane 4: DNA sample from Lane 2
after ligation; Lane 5: 1 kb DNA ladder. DNA samples were electrophoresed in 1%
agarose gel. (B) DNA sequencing confirmations of the cloning sites.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
OriginalVector
ReconstitutedVector
OHLeft Ins right Ins
Insert
ReconstitutedVector
OriginalVector
ReconstitutedVector
OriginalVector
ReconstitutedVector
Insert
A B C D
OriginalVector
1st PCR
2nd PCR
Annealing
Ligation
1st PCR
2nd PCR
Annealing
Ligation
1st PCR
2nd PCR
Annealing
Ligation
1st PCR
2nd PCR
Annealing
Ligation
Fw2
Rv1
Left Ins
Right Ins
OH
Rv2
Fw1
Fw1Left OH Fw2
Fw2Fw1 Right OH
Insert
Fw2
Rv1Del
OH
Fw1
Rv2OH
Fw2
Rv1
Fw1
Rv2
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was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
B C
D E
A
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Fw1
Rv5
12
3
4
5
1
OriginalVector
Fw21st PCR Fw5/Rv5
2
Fw3
Rv2 3
Fw4
Rv3 4
Fw5
Rv4 5Rv1
1st PCR Fw1/Rv1
1st PCR Fw2/Rv2
1st PCR Fw3/Rv3
1st PCR Fw4/Rv4
2nd PCR Fw5/Rv5
2nd PCR Fw1/Rv1
2nd PCR Fw2/Rv2
2nd PCR Fw3/Rv3
2nd PCR Fw4/Rv4
12
3
4
5
OH4 OH5OH3OH2OH1
ReconstitutedVector
A
B CQ70A Q209A Q342A
D463A Q597A R732A
OH Overhang
Mutant
Annealing
Ligationwas not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
OriginalVector1
OriginalVector2
OriginalVector3
ReconstitutedVector
1 2 3 4 5 6 7 8 9A B
CT4L 5' cloning site T4L 3' and MCM6 5'
cloning site
MCM6 3' cloning site
OH1
OH2
OH3
OH1: Overhang 1
OH2: Overhang 2
OH3: Overhang 3
Gene 1 Gene 2
Annealing
Ligation
Annealing Annealing
1st PCR Fw1/Rv1
2nd PCR Fw2/Rv2
1st PCR Fw1/Rv1
2nd PCR Fw2/Rv2
1st PCR Fw1/Rv1
2nd PCR Fw2/Rv2
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1 2 3 4 5Q32A
A B
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1
Supplementary methods
Optimization of key parameters for AFEAP cloning. For the experiment of optimizing
overhang sizes for efficient assembly, we designed a set of special linear DNAs with varying
size from 5.5 to 30 kb (total five different DNAs), but each containing same sequences at 5'
(5'CTAACTACTTGCTCGAAGATTGAG3') and 3'
(5'TTCTGCAGATATCCAGCACAGTGG3') terminal for convenient primer designing
(Figure 1c). Primers were designed to add 0 to 20 bp (total nine different overhangs) overhang
adapter sequence at 5' ends of DNA molecules. For each test, we performed AFEAP cloning
procedures as shown at Figure 1a. PCR conditions were listed in Supplementary Table S3, and
thermocycling conditions were listed in the Supplementary Table S4. According, step1: five
PCRs were performed to generate five double-stranded DNA fragments used primer pairs
OHtestfw1/OHtestrv1 and 5.5 kb, 8.0 kb, 15 kb, 20 kb, or 30 kb DNA as template. PCR products
were gel purified. Step 2: Two single-primer PCRs in parallel were performed to generate two
complementary single-stranded DNA fragments using each purified fragment generated in the
Step 1 as template and single primer of OHtest1fw2 or OHtest1rv2, OHtest2fw2 or OHtest2rv2,
OHtest3fw2 or OHtest3rv2, OHtest4fw2 or OHtest4rv2, OHtest5fw2 or OHtest5rv2, OHtest8fw2
or OHtest8rv2, OHtest10fw2 or OHtest10rv2, OHtest14fw2 or OHtest14rv2, or OHtest20fw2 or
OHtest20rv2 (for detailed primer sequences see Supplementary Table S2). Step 3: The newly
synthesized complementary PCR products in step 2 were annealed using the conditions as shown
in Supplementary Table S5. Step 4: total 1 µg of annealed DNAs with nick were sealed by T4
DNA ligase (NEB) to form transformable plasmid used protocol from NEB. Reconstituted vector
was transformed into competent E.coli cells. The colonies forming were counted, and the join
sites were confirmed by DNA sequence (Supplementary Figure S1a-g).
To determine the effect of the 5' end of the overhang as G/C or A/T on the efficiency of
assembly with AFEAP method, we designed four primers: OHtestGCfw2, OHtestGCrv2,
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2
OHtestATfw2, and OHtestATrv2 (See Supplementary Table S2). We ran AFEAP cloning as
shown at Figure 1a. PCR conditions were listed in Supplementary Table S3, and thermocycling
conditions were listed in the Supplementary Table S4. According, step1: 5 PCRs were
performed to generate five double-stranded DNA fragments used primer pairs: OHtestfw1 and
OHtestrv1, and 5.5 kb, 8.0 kb, 15 kb, 20 kb, or 30 kb DNA fragments as templates. PCR products
were gel purified. Step 2: Two single-primer PCRs in parallel were performed to generate two
complementary single-stranded DNA fragments using each purified fragment generated in the
Step 1 as template and single primer of OHtestGCfw2 or OHtestGCrv2, or OHtestATfw2 or
OHtestATrv2. Step 3: The newly synthesized complementary single-stranded DNA fragments
were annealed using the conditions as shown in Supplementary Table S5. Step 4: total 1 µg of
annealed DNAs with nick were sealed by T4 DNA ligase (NEB) to form transformable plasmid
used protocol from NEB. Reconstituted vector was transformed into competent E.coli cells. The
colonies were counted, and the join sites were confirmed by DNA sequencing (Supplementary
Figure S1h and i).
To determine the effect of ligation on the assembly, we ran AFEAP cloning as shown at
Figure 1a. According, step1: Five PCRs were performed to generate five double-stranded DNA
fragments used primer pairs: OHtestfw1 and OHtestrv1, and 5.5 kb, 8.0 kb, 15 kb, 20 kb, or 30 kb
DNA fragments as templates. PCR products were gel purified. Step 2: Two single-primer PCRs
in parallel were performed to generate two complementary single-stranded DNA fragments using
each purified fragment generated in the Step 1 as template and single primer of OHtest5fw2 or
OHtest5rv2. Step 3: The newly synthesized complementary single-stranded DNA fragments were
annealed using the conditions as shown in Supplementary Table S5. Step 4: The annealed
products were treated or never treated with T4 DNA ligase, and same amount of DNAs were
transformed into competent E.coli cells. The colonies were counted, and the join sites were
confirmed by DNA sequencing.
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3
Assembly of multiple fragments with AFEAP cloning. To evaluate the effect of fragment
number on assembly efficiency, we built a pET22b-FLAG-T4L-GGSGGlinker-MCM6 tandem
construct, encoding T4 lysozyme (T4L)12 and MCM6 protein fused by a peptide linker, from
varying number of DNA fragments (Figure 2a) with AFEAP cloning method. PCR products
were subjected to AFEAP cloning protocol as mentioned above (Figure 1a). 11 unique
conditions (assemblies of 2+V to 12+V fragments) were designed and tested (Figure 2a). For
each test, we performed AFEAP cloning procedures as shown at Figure 1a. PCR reactions were
listed in Supplementary Table S3, and thermocycling conditions were listed in the
Supplementary Table S4. Here we used the assembly of 2+V fragments as example. Step 1:
three PCRs were performed to generate three double-stranded DNA fragments used primer pairs
8Site13fw1 and 8Site1rv1, 8Site1fw1 and 8Site3rv1, or 8Site3fw1 and 8Site13rv1, and pET22b,
the DNA sequence that encodes T4 lysozyme, or E.coli genome DNA as templates. PCR products
were gel purified. Step 2: Two single-primer PCRs in parallel were performed to generate two
complementary single-stranded DNA fragments using each purified fragment generated in the
Step 1 as template and single primer of 8Site13fw2 or 8Site1rv2, 8Site1fw2 or 8Site3rv21, and
8Site3fw2 or 8Site13rv2. Step 3: The newly synthesized complementary single-stranded DNA
fragments were annealed using the conditions as shown in Supplementary Table S5 to produce
double-stranded DNAs with sticky ends. Step 4: The DNA fragments with complementary
sticky ends were cycled ligation assembly by T4 ligase (NEB). DNA ligation reactions were
performed to fuse DNA fragments 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 reaction was incubated at room temperature for 2 hours.
After heat inactivation at 65°C for 10 min, the reaction was chilled on ice. Reconstituted vector
was transformed into competent E.coli cells. The colonies forming were counted, and the join
sites can be confirmed by DNA sequencing.
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4
Assembly of BAC with AFEAP cloning. A bacterial artificial chromosome (BAC), which
contains 200 kb DNA sequence insert, was constructed with AFEAP cloning. PCR products
were subjected to AFEAP cloning protocol as mentioned above (Figure 1a). PCR reactions were
listed in Supplementary Table S3, and thermocycling conditions were listed in the
Supplementary Table S4. Step 1: 9 PCRs were performed to generate 9 double-stranded DNA
fragments used primer pairs BACSite1fw1 and BACSite2rv1, BACSite2fw1 and BACSite3rv1,
BACSite3fw1 and BACSite4rv1, BACSite4fw1 and BACSite5rv1, BACSite5fw1 and
BACSite6rv1, BACSite6fw1 and BACSite7rv1, BACSite7fw1 and BACSite8rv1, BACSite8fw1
and BACSite9rv1, or BACSite9fw1 and BACSite1rv1, and pCC1BACTM vector or genome of
Streptomyces albus subsp. albus as templates. PCR products were gel purified. Step 2: Two
single-primer PCRs in parallel were performed to generate two complementary single-stranded
DNA fragments using each purified fragment generated in the Step 1 as template and single
primer of BACSite1fw2 or BACSite2rv2, BACSite2fw2 or BACSite3rv2, BACSite3fw2 or
BACSite4rv2, BACSite4fw2 or BACSite5rv2, BACSite5fw2 or BACSite6rv2, BACSite6fw2 or
BACSite7rv2, BACSite7fw2 or BACSite8rv2, BACSite8fw2 or BACSite9rv2, or BACSite9fw2
or BACSite1rv2. Step 3: The newly synthesized complementary single-stranded DNA fragments
were annealed using the conditions as shown in Supplementary Table S5 to generate double-
stranded DNAs with sticky ends. Step 4: The DNA fragments with complementary sticky ends
were cycled ligation assembly by T4 ligase (NEB). DNA ligation reactions were performed to
fuse DNA fragments 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 1:1:1:1:1:1:1:1:10. The reaction was incubated at room temperature for 2
hours. After heat inactivation at 65 °C for 10 min, the reaction was chilled on ice. Step 5:
Electroporation was carried out to transform constructed BAC into electrocompetent cells. The
colonies forming were counted, and the join sites can be confirmed by DNA sequencing.
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5
Supplementary references
1. Dagert, M.; Ehrlich, S. D., Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene 1979, 6 (1), 23-8. 2. Jin, P.; Ding, W.; Du, G.; Chen, J.; Kang, Z., DATEL: A Scarless and Sequence-Independent DNA Assembly Method Using Thermostable Exonucleases and Ligase. ACS synthetic biology 2016, 5 (9), 1028-32. 3. Quan, J.; Tian, J., Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One 2009, 4 (7), e6441. 4. Li, M. Z.; Elledge, S. J., Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods 2007, 4 (3), 251-6. 5. Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.; Hutchison, C. A., 3rd; Smith, H. O., Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 2009, 6 (5), 343-5. 6. Bitinaite, J.; Rubino, M.; Varma, K. H.; Schildkraut, I.; Vaisvila, R.; Vaiskunaite, R., USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Research 2007, 35 (6), 1992-2002. 7. Engler, C.; Gruetzner, R.; Kandzia, R.; Marillonnet, S., Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One 2009, 4 (5), e5553. 8. de Kok, S.; Stanton, L. H.; Slaby, T.; Durot, M.; Holmes, V. F.; Patel, K. G.; Platt, D.; Shapland, E. B.; Serber, Z.; Dean, J.; Newman, J. D.; Chandran, S. S., Rapid and reliable DNA assembly via ligase cycling reaction. ACS synthetic biology 2014, 3 (2), 97-106. 9. Shao, Z.; Zhao, H., DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Research 2009, 37 (2), e16. 10. Liu, C. J.; Jiang, H.; Wu, L.; Zhu, L. Y.; Meng, E.; Zhang, D. Y., OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments. Scientific reports 2017, 7, 44648. 11. Liang, J.; Liu, Z.; Low, X. Z.; Ang, E. L.; Zhao, H., Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method. Nucleic Acids Research 2017. 12. Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen, S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis, W. I.; Kobilka, B. K.; Stevens, R. C., High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science 2007, 318 (5854), 1258-65.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
a b c d
f g h i
e
Supplementary Figure S1. Sequencing validation of assemble with various overhangs. (a)-(g) various
overhang sizes; (h) overhang designed as 5' end of G/C; (i) overhang designed as 5' end of A/T. Overhang
regions were marked by red dashed line rectangles.
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S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 S13
12+V
11+V
10+V
9+V
8+V
7+V
6 +V
5+V
4+V
3+V
2+V
Supplementary Figure S2. Sequencing validation of number of fragments characterization. The join
sites were shown as S1 to S13, and number of fragments for assembly was shown as 2+V to 12+V. The
overhang sequences were shown. V: vector backbone.
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a
b
c
d
S1 S2 S3 S4 S5 S6 S7
Supplementary Figure S3. Sequencing validation of plasmid sizes characterization. Five join sites are
S1, S2, S3, S4, and S5. (a) 11.5 kb plasmid; (b) 19.6 kb plasmid; (c) 28 kb plasmid; (d) 34.6 kb plasmid.
The overhang sequences were shown.
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Supplementary Table S1 Comparisons of AFEAP cloning with common DNA assembly methods
Capabilitya Scarless Step(s) Reference
DATEL 2−10 DNA fragments with fidelity between 74 and
100% yes 1 2
CPEC 9 kb plasmid from 5 fragments at ∼90% fidelity yes 1 3
SLIC 8 kb plasmid from 10 fragments at ∼20% fidelity yes 2 4
Gibson Up to several hundred kilobases, but did not enable assembly of more than four DNA parts with more
than 50% of clones being correct. yes 1 5
USER 8 kb plasmid from 11 fragments at ∼60% fidelity yes 1 6
Golden At least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones
obtained containing the desired construct. yes 1 7
LCR up to 12 DNA parts with 60–100% of individual clones being correct yes 1 8
DNA assembler ∼9 kb DNA consisting of three genes, ∼11 kb DNA consisting of five genes, and ∼19 kb consisting of
eight genes with high efficiencies (70–100%) yes 3 9
OEPR Large DNA fragments up to 6 kb or multiple DNA fragments up to two 3 kb, three 2 kb and four 1 kb
into vectors (8 kb tested). yes 1 10
TPA 7 kb plasmid from 10 fragments at ∼80% fidelity and 31 kb plasmid from five fragments at ∼50%
fidelity. yes 1 11
AFEAP 8 kb plasmid from 13 fragments at ∼80% fidelity and 35.6 kb plasmid from six fragments at ∼82%
fidelity, 200 kb plasmid from 9 fragments at ∼47%. yes 1 This study
a The largest demonstrated number of fragments, plasmids size, and fidelity are reported in references.
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Supplementary Table S2 Primers used in this work
Name Primers (5'→3')*
Various length of overhangs OHtestfw1 TATTCTGCAGATATCCAGCACAGTGG OHtestrv1 CTCAATCTTCGAGCAAGTAGTTAG
OHtest2fw2 AATATTCTGCAGATATCCAGCACAGTGG OHtest2rv2 TTCTCAATCTTCGAGCAAGTAGTTAG OHtest3fw2 GAATATTCTGCAGATATCCAGCACAGTGG OHtest3rv2 TTCCTCAATCTTCGAGCAAGTAGTTAG OHtest4fw2 AGAATATTCTGCAGATATCCAGCACAGTGG OHtest4rv2 TTCTCTCAATCTTCGAGCAAGTAGTTAG OHtest5fw2 GAGAATATTCTGCAGATATCCAGCACAGTGG OHtest5rv2 TTCTCCTCAATCTTCGAGCAAGTAGTTAG OHtest8fw2 ATTGAGAATATTCTGCAGATATCCAGCACAGTGG OHtest8rv2 TTCTCAATCTCAATCTTCGAGCAAGTAGTTAG
OHtest10fw2 AGATTGAGAATATTCTGCAGATATCCAGCACAGTGG OHtest10rv2 TTCTCAATCTCTCAATCTTCGAGCAAGTAGTTAG OHtest14fw2 TCGAAGATTGAGAATATTCTGCAGATATCCAGCACAGTGG OHtest14rv2 TTCTCAATCTTCGACTCAATCTTCGAGCAAGTAGTTAG OHtest20fw2 CATTGCTCGAAGATTGAGAATATTCTGCAGATATCCAGCACAGTGG OHtest20rv2 TTCTCAATCTTCGAGCAATGCTCAATCTTCGAGCAAGTAGTTAG
Analysis of the effect of 5’ end of the overhang OHtestGCfw2 GTTGAGACTATTCTGCAGATATCCAGCACAGTGG OHtestGCrv2 CTCTCAAGCTCAATCTTCGAGCAAGTAGTTAG OHtestATfw2 ATTGAGATTATTCTGCAGATATCCAGCACAGTGG OHtestATrv2 ATCTCAATCTCAATCTTCGAGCAAGTAGTTAG
Assembly of 8 kb plasmid Primers for the assembly of site 1
8Site1fw1 GACTACAAGGATGAAGAGGACAAGAACATCTTTGAAATGCTGCGTATTG 8Site1rv1 TATATCTCCTTCTTAAAGTTAAAC 8Site1fw2 CATATGGACTACAAGGATGAAGAGGACAAGAAC 8Site1rv2 CATATGTATATCTCCTTCTTAAAGTTAAAC
Primers for the assembly of site 2 8Site2fw1 GTGGTATTCTGCGCAATGCAAAAC 8Site2rv1 TGCGTCCACATCCTGGTTAAAC 8Site2fw2 GCTGTTCTGCGTCCACATCCTGGTTAAAC 8Site2rv2 GAACAGCCACCACCACCACCACCACTGAGATC
Primers for the assembly of site 3
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8Site3fw1 GGCGGATCAGGCGGTGACCTCGCGGCGGCAGCGGAGCC 8Site3rv1 GTCCCAGGTGCCGGTGCGAAAGGTAATTG 8Site3fw2 GCCTATGGCGGATCAGGCGGTGACCTCG 8Site3rv2 ATAGGCGTCCCAGGTGCCGGTGCGAAAGGTAATTG
Primers for the assembly of site 4 8Site4fw1 GAGTTCTATAGAGTTTACCCTTAC 8Site4rv1 AATGGTGGTGGAAAGTTGCTGG 8Site4fw2 CAAGAGGAGTTCTATAGAGTTTACCCTTAC 8Site4rv2 CTCTTGAATGGTGGTGGAAAGTTGCTGG
Primers for the assembly of site 5 8Site5fw1 GATGTAGAACAGCAGTTCAAATAC 8Site5rv1 ACTGTCTGACAGTCCAAGCAC 8Site5fw2 GATCAGGGATGTAGAACAGCAGTTCAAATAC 8Site5rv2 CCTGATCACTGTCTGACAGTCCAAGCAC
Primers for the assembly of site 6 8Site6fw1 CCTGACGTCTCCAAGCTTAGCAC 8Site6rv1 AATCAGTGTCCCTGTAAAGTCAC 8Site6fw2 GTTGTGCCTGACGTCTCCAAGCTTAGCAC 8Site6rv2 CACAACAATCAGTGTCCCTGTAAAGTCAC
Primers for the assembly of site 7 8Site7fw1 GAGAAAGTGTTTGAGATGAGTC 8Site7rv1 TTTCACAGTCATTTGGTTCTTAATG 8Site7fw2 GAATGGGAGAAAGTGTTTGAGATGAGTC 8Site7rv2 CCATTCTTTCACAGTCATTTGGTTCTTAATG
Primers for the assembly of site 8 8Site8fw1 CCAGAGCTGTCTACACCAGTGG 8Site8rv1 AACTCCTCCACGTGCTTGAGAAATTG 8Site8fw2 CAGCCCCAGAGCTGTCTACACCAGTGG 8Site8rv2 GGCTGAACTCCTCCACGTGCTTGAGAAATTG
Primers for the assembly of site 9 8Site9fw1 CATTTTGGCAGCAGCAAACCCAATC 8Site9rv1 CGGGCGTTCAGAGTAGCCTTCAC 8Site9fw2 GACGTCCATTTTGGCAGCAGCAAACCCAATC 8Site9rv2 GACGTCCGGGCGTTCAGAGTAGCCTTCAC
Primers for the assembly of site 10 8Site10fw1 CAAGACAGTTTAAACCCAAGATTTC 8Site10rv1 AGAAGATATCTTCTGATATCATC 8Site10fw2 CTTTGCAAGACAGTTTAAACCCAAGATTTC 8Site10rv2 CAAAGAGAAGATATCTTCTGATATCATC
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Primers for the assembly of site 11 8Site11fw1 GTGTGGAAACACCTGATGTCAATC 8Site11rv1 GATTGACATCAGGTGTTTCCACAC 8Site11fw2 CATCCGTGTGGAAACACCTGATGTCAATC 8Site11rv2 GGATGGATTGACATCAGGTGTTTCCACAC
Primers for the assembly of site 12 8Site12fw1 GAGTCAGCATTAAAGAGGAGCG 8Site12rv1 TTCTTCTTCCACCTTTCTGAGG 8Site12fw2 GAGGACGAGTCAGCATTAAAGAGGAGCG 8Site12rv2 GTCCTCTTCTTCTTCCACCTTTCTGAGG
Primers for the assembly of site 13 8Site13fw1 CACCACCACCACCACCACTGAGATC 8Site13rv1 ATCTTCGAGCAAGTAGTTAGGG 8Site13fw2 CTCGAGCACCACCACCACCACCACTGAGATC 8Site13rv2 CTCGAGATCTTCGAGCAAGTAGTTAGGG
Assembly of 11.5 kb plasmid Primers for the assembly of site 1
11.5Site1fw1 ACCGGTCACCCGGTATTCCATTC 11.5Site1rv1 TATATCTCCTTCTTAAAGTTAAAC 11.5Site1fw2 CATATGACCGGTCACCCGGTATTCCATTC 11.5Site1rv2 CATATGTATATCTCCTTCTTAAAGTTAAAC
Primers for the assembly of site 2 11.5Site2fw1 CTGCTGCGGGCCCTGCTCGAAG 11.5Site2rv1 GTTGCCCCCGGTGACGGTCAG 11.5Site2fw2 CCGCTGCTGCTGCGGGCCCTGCTCGAAG 11.5Site2rv2 CAGCGGGTTGCCCCCGGTGACGGTCAG
Primers for the assembly of site 3 11.5Site3fw1 CCGGAAACGGCACCAGCGAGGC 11.5Site3rv1 CCCACAAGGCCGCTGTCGGCTG 11.5Site3fw2 CCCTGCCCGGAAACGGCACCAGCGAGGC 11.5Site3rv2 GCAGGGCCCACAAGGCCGCTGTCGGCTG
Primers for the assembly of site 4 11.5Site4fw1 CGGGCGGCAACACCAACCGTGAG 11.5Site4rv1 TGTCGCGACTCGCATCTCGGACTC 11.5Site4fw2 CTGGCGGCGGGCGGCAACACCAACCGTGAG 11.5Site4rv2 CCGCCAGTGTCGCGACTCGCATCTCGGACTC
Primers for the assembly of site 5 11.5Site5fw1 GGAAAGCAGGCACGGTGTTCC 11.5Site5rv1 GGAATTTCATCGTGGCGGATC
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11.5Site5fw2 CCTTTTCGGAAAGCAGGCACGGTGTTCC 11.5Site5rv2 GAAAAGGGGAATTTCATCGTGGCGGATC
Primers for the assembly of site 6 11.5Site6fw1 CGCGTACTCGTCGAGGTGCTCGTTC 11.5Site6rv1 TTCGGGATCGGCGTCGCGGAG 11.5Site6fw2 GGCCGCCGCGTACTCGTCGAGGTGCTCGTTC 11.5Site6rv2 GCGGCCTTCGGGATCGGCGTCGCGGAG
Primers for the assembly of site 7 11.5Site7fw1 CACCACCACCACCACCACTGAGATC 11.5Site7rv1 CTGACGGCCGGGCATCAATGTC 11.5Site7fw2 CTCGAGCACCACCACCACCACCACTGAGATC 11.5Site7rv2 CTCGAGCTGACGGCCGGGCATCAATGTC
Assembly of 19.6 kb plasmid Primers for the assembly of site 1
19.6Site1fw1 GACCCCAGCTGGACGACCCGCAG 19.6Site1rv1 TATATCTCCTTCTTAAAGTTAAAC 19.6Site1fw2 CATATGGACCCCAGCTGGACGACCCGCAG 19.6Site1rv2 CATATGTATATCTCCTTCTTAAAGTTAAAC
Primers for the assembly of site 2 19.6Site2fw1 GGCGAACGACCCGTATGCGATG 19.6Site2rv1 GGGTTGGGATGCGGGTACTCTTC 19.6Site2fw2 CTGTTCCGGCGAACGACCCGTATGCGATG 19.6Site2rv2 GGAACAGGGGTTGGGATGCGGGTACTCTTC
Primers for the assembly of site 3 19.6Site3fw1 CATCGGGGTGGTGGCCCCCGG 19.6Site3rv1 ATCACGACCCGCCGGGTCATC 19.6Site3fw2 CACCGGCATCGGGGTGGTGGCCCCCGG 19.6Site3rv2 CCGGTGATCACGACCCGCCGGGTCATC
Primers for the assembly of site 4 19.6Site4fw1 TCCGGCACGGCGGCCCTGGTCC 19.6Site4rv1 CCGGGGCCGTCCGAGGACGAGG 19.6Site4fw2 CCGGTCTCCGGCACGGCGGCCCTGGTCC 19.6Site4rv2 GACCGGCCGGGGCCGTCCGAGGACGAGG
Primers for the assembly of site 5 19.6Site5fw1 CGATCAGACCAACAGCGAGTTGG 19.6Site5rv1 ATGACACCGTCCTTGGGAGAAG 19.6Site5fw2 GATCAGGCGATCAGACCAACAGCGAGTTGG 19.6Site5rv2 CCTGATCATGACACCGTCCTTGGGAGAAG
Primers for the assembly of site 6
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19.6Site6fw1 GGCGGGGCTGCACCTGCACTCCG 19.6Site6rv1 TCCTCCACCCGCTGCGCGCGG 19.6Site6fw2 GCTGGGGGCGGGGCTGCACCTGCACTCCG 19.6Site6rv2 CCCAGCTCCTCCACCCGCTGCGCGCGG
Primers for the assembly of site 7 19.6Site7fw1 CACCACCACCACCACCACTGAGATC 19.6Site7rv1 GCGGTCATGACTGGGCGACCTC 19.6Site7fw2 CTCGAGCACCACCACCACCACCACTGAGATC 19.6Site7rv2 CTCGAGGCGGTCATGACTGGGCGACCTC
Assembly of 28 kb plasmid Primers for the assembly of site 1
28Site1fw1 GCCGACACTCCCGCCTCGGACAAACG 28Site1rv1 TATATCTCCTTCTTAAAGTTAAAC 28Site1fw2 CATATGGCCGACACTCCCGCCTCGGACAAACG 28Site1rv2 CATATGTATATCTCCTTCTTAAAGTTAAAC
Primers for the assembly of site 2 28Site2fw1 GTGAGCGGCAGCATCGGGCACCCCTG 28Site2rv1 GGCCAAAGGCACGCAGCCTGGGG 28Site2fw2 CGTGACGTGAGCGGCAGCATCGGGCACCCCTG 28Site2rv2 GTCACGGGCCAAAGGCACGCAGCCTGGG
Primers for the assembly of site 3 28Site3fw1 CGGTGCGCGGCGAACTTGAGC 28Site3rv1 CCGCTACACCCCGCGCTGCGTGC 28Site3fw2 CGCAGGCGGTGCGCGGCGAACTTGAGC 28Site3rv2 CCTGCGCCGCTACACCCCGCGCTGCGTGC
Primers for the assembly of site 4 28Site4fw1 GTCCTCGCGCGCGGCGCCGGCGGC 28Site4rv1 CTCGACAGGGGGCACGTGCGTG 28Site4fw2 GGCCCTCGTCCTCGCGCGCGGCGCCGGCGGC 28Site4rv2 GAGGGCCCTCGACAGGGGGCACGTGCGTG
Primers for the assembly of site 5 28Site5fw1 CTGGAGACCATCGAACGAGTACG 28Site5rv1 TGCGGTGCCGGAAACCCGGG 28Site5fw2 GATCAGGCTGGAGACCATCGAACGAGTACG 28Site5rv2 CCTGATCTGCGGTGCCGGAAACCCGGG
Primers for the assembly of site 6 28Site6fw1 GAAACTGCCCGAGCCCAGCGGG 28Site6rv1 AGGGTCGCCACCGAGCCGGTGG 28Site6fw2 GGCCCGGAAACTGCCCGAGCCCAGCGGG
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28Site6rv2 CGGGCCAGGGTCGCCACCGAGCCGGTGG Primers for the assembly of site 7
28Site7fw1 CACCACCACCACCACCACTGAGATC 28Site7rv1 GGAATTTCAATGATCCTTGG 28Site7fw2 CTCGAGCACCACCACCACCACCACTGAGATC 28Site7rv2 CTCGAGGGAATTTCAATGATCCTTGG
Assembly of 35.6 kb plasmid Primers for the assembly of site 1
35.6Site1fw1 GATCAGCTCGTCCCGTTCGGAG 35.6Site1rv1 TATATCTCCTTCTTAAAGTTAAAC 35.6Site1fw2 CATATGGATCAGCTCGTCCCGTTCGGAG 35.6Site1rv2 CATATGTATATCTCCTTCTTAAAGTTAAAC
Primers for the assembly of site 2 35.6Site2fw1 CCGCTGGAGATCGTGCCGTTCG 35.6Site2rv1 GGGGACGAGCGTGTCGTAGTCG 35.6Site2fw2 GACTACCCGCTGGAGATCGTGCCGTTCG 35.6Site2rv2 GTAGTCGGGGACGAGCGTGTCGTAGTCG
Primers for the assembly of site 3 35.6Site3fw1 CCGGCCGCCGGTGACGCCGCTG 35.6Site3rv1 GCGAGGACGACGACGTCGGGCGCCGTG 35.6Site3fw2 GGAGCTGCCGGCCGCCGGTGACGCCGCTG 35.6Site3rv2 CAGCTCCGCGAGGACGACGACGTCGGGCGCCGTG
Primers for the assembly of site 4 35.6Site4fw1 GGCGTCGCCGGGCCGGACGCCC 35.6Site4rv1 TGCCGCCAGGTGATCTCGCCGG 35.6Site4fw2 CGAGCTCGGCGTCGCCGGGCCGGACGCCC 35.6Site4rv2 GAGCTCGTGCCGCCAGGTGATCTCGCCGG
Primers for the assembly of site 5 35.6Site5fw1 CCGGTTCATCGAGATGGGCAAG 35.6Site5rv1 GGCCGCACCAGGCGCAGCGAG 35.6Site5fw2 GGGCGGCCGGTTCATCGAGATGGGCAAG 35.6Site5rv2 CCGCCCGGCCGCACCAGGCGCAGCGAG
Primers for the assembly of site 6 35.6Site6fw1 CAGTTCGACCCGCTCCTCTTC 35.6Site6rv1 GTCGCGCAGGAAGCCGCCGCGGTTG 35.6Site6fw2 GCCGACCAGTTCGACCCGCTCCTCTTC 35.6Site6rv2 GTCGGCGTCGCGCAGGAAGCCGCCGCGGTTG
Primers for the assembly of site 7 35.6Site7fw1 CACCACCACCACCACCACTGAGATC
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
35.6Site7rv1 TCAGTCAGTCGTCCAGGCGCGCCAG 35.6Site7fw2 CTCGAGCACCACCACCACCACCACTGAGATC 35.6Site7rv2 CTCGAGTCAGTCAGTCGTCCAGGCGCGCCAG
200 kb BAC assembly Primers for the assembly of site 1
BACSite1fw1 GGATGCGAAGGACGCGCTGCGCAAGG BACSite1rv1 CCGGGTACCGAGCTCGAATTCGC BACSite1fw2 GGATCCGGATGCGAAGGACGCGCTGCGCAAGG BACSite1rv2 GGATCCCCGGGTACCGAGCTCGAATTCGC
Primers for the assembly of site 2 BACSite2fw1 CGGTACGCCCCCGTGGTGATCACCG BACSite2rv1 AGACGGCCTGCCCGGAGCCGGCGAG BACSite2fw2 GCGACCGCCGGTACGCCCCCGTGGTGATCACCG BACSite2rv2 GCGGTCGCAGACGGCCTGCCCGGAGCCGGCGAG
Primers for the assembly of site 3 BACSite3fw1 CCGCCGAACGCGGCCACGAGGTCAC BACSite3rv1 GCGCAGGCGAGCCCGGCGGGAC BACSite3fw2 CGTCTCCGCCGCCGAACGCGGCCACGAGGTCAC BACSite3rv2 CGGAGACGGCGCAGGCGAGCCCGGCGGGAC
Primers for the assembly of site 4 BACSite4fw1 GAGCTCGCTGCCTTGACCCCCCG BACSite4rv1 GCCGGGCTCAGCCGCTGCCGAG BACSite4fw2 GGGGCGAGGAGCTCGCTGCCTTGACCCCCCG BACSite4rv2 CTCGCCCCGCCGGGCTCAGCCGCTGCCGAG
Primers for the assembly of site 5 BACSite5fw1 CAGGCTGCGCTGGATCTGCAGCGAG BACSite5rv1 CTTCCACGAGGAGTCGCTCGCCC BACSite5fw2 GCCGCGTGCAGGCTGCGCTGGATCTGCAGCGAG BACSite5rv2 CACGCGGCCTTCCACGAGGAGTCGCTCGCCC
Primers for the assembly of site 6 BACSite6fw1 GCGGGTGGCGCGATGGTCTCCATAC BACSite6rv1 CTCCATCAACCGACCACGAGCAG BACSite6fw2 GCGCTTCCCGCGGGTGGCGCGATGGTCTCCATAC BACSite6rv2 GGGAAGCGCCTCCATCAACCGACCACGAGCAG
Primers for the assembly of site 7 BACSite7fw1 GCTCGACGTGGACGCGGTCGAGGCC BACSite7rv1 GGGCGTTGGCGAGGGCCTGGCGGATC BACSite7fw2 GCCTCTCCGCGCTCGACGTGGACGCGGTCGAGGCC BACSite7rv2 GCGGAGAGGCGGGCGTTGGCGAGGGCCTGGCGGATC
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Primers for the assembly of site 8 BACSite8fw1 CGCCGGTATCGACCCGGGCACCCTCAAG BACSite8rv1 TCTCCCAGGCCGATTCGAGCAGCAGC BACSite8fw2 CCTTCGAACGCGCCGGTATCGACCCGGGCACCCTCAAG BACSite8rv2 CGTTCGAAGGTCTCCCAGGCCGATTCGAGCAGCAGC
Primers for the assembly of site 9 BACSite9fw1 GAGTATTCTATAGTCTCACCTAAATAG BACSite9rv1 TTGCGTGCCGCCGCCGCGGCGC BACSite9fw2 AAGCTTGAGTATTCTATAGTCTCACCTAAATAG BACSite9rv2 AAGCTTTTGCGTGCCGCCGCCGCGGCGC
* Bold purple and green letters show overhang sequences.
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint
Supplementary Table S3 PCR conditions
Reaction 1 Reaction 2a Reaction 2b Template DNA ~50 ng ~500 ng ~500 ng
Forward primer (100 µM) 0.25 µL 0.25 µL Reverse primer (100 µM) 0.25 µL 0.25 µL Phusion GC Buffer (5×) 10 µL 10 µL 10 µL
dNTPs (10 mM) 1 µL 1 µL 1 µL DMSO (100%) 1.5 µL 1.5 µL 1.5 µL
Phusion High Fidelity DNA Polymerase
1 µL 1 µL 1 µL
add water to 50 µL 50 µL 50 µL
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Supplementary Table S4 PCR thermocycling conditions
Step Temperature Time
Initial Denaturation 98°C 5 min
20 Cycles
98°C 20 seconds 60-50 °C,
step -0.5 °C 20 seconds
72°C 1 minute/kbp 98°C 20 seconds 10 Cycles 52 °C 20 seconds 72°C 1 minute/kbp
Final Extension 72°C 10 minutes
Hold 4 °C
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Supplementary Table S5 PCR product reannealing conditions
Steps Temperature (°C) Time (min) 1 95 5 2 90 1 3 80 1 4 70 0.5 5 60 0.5 6 50 0.5 7 40 0.5 8 37 Holding
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted May 17, 2020. . https://doi.org/10.1101/2020.05.16.100107doi: bioRxiv preprint