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

was not certified by peer review) is the author/funder.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

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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


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


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


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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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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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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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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.


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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.


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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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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.


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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


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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.


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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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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

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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

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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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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



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



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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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.


https://doi.org/10.1101/2020.05.16.100107

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


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


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


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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.


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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


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a simple and efficient method for in vitro site-directed ...site-directed mutagenesis, which...

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