Bhat et al.

Virology Journal (2020) 17:82

https://doi.org/10.1186/s12985-020-01358-2

METHODOLOGY Open Access

A ligation and restriction enzyme

independent cloning technique: an
alternative to conventional methods for
cloning hard-to-clone gene segments in
the influenza reverse genetics system
Sushant Bhat, Dagmara Bialy, Joshua E. Sealy, Jean-Remy Sadeyen, Pengxiang Chang and Munir Iqbal*

Abstract
Background: Reverse genetics is used in many laboratories around the world and enables the creation of tailor-
made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties
remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS).
Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-
directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant
plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of
internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning
process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing
plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to
erroneous incorporation of bacterial genomic sequences into the influenza gene of interest.
Methods: Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method
for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers
followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation.
Results: Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/
23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014
(H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued.
Conclusion: The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which
have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in
bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be
applied to clone any influenza gene segment using universal primers, which would help in rapid generation of
influenza viruses and facilitate influenza research and vaccine development.
Keywords: Influenza, Reverse genetics, Polymerase, Restriction enzyme independent cloning

* Correspondence: [email protected]
The Pirbright Institute, Surrey, UK

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Bhat et al. Virology Journal (2020) 17:82 Page 2 of 9

Background (MCS) followed by 12 conserved nucleotide bases of in-

Reverse Genetics (RG) is the process of in vitro generation fluenza untranslated region (UTR) and 2–6 segment spe-
of live virus with synthetic or PCR amplified genes [1]. This cific nucleotides towards the 3′ end. The reverse primer
technique enables the creation of mutant influenza viruses constitutes 13 bases complementary to the other end of
of any desired genotype or phenotype. The RG technique the pHW2000 MCS followed by influenza UTR, which
was first employed for the rabies virus in the year 1994 [2], contains the conserved 13 bases followed by 5–6 seg-
and this was soon followed by the establishment of the ment specific bases at the 3′ end. The primer sequences
in vitro generation of a range of DNA and RNA viruses (in- for cloning PB2 and PB1 are described in Table 1. The
cluding segmented or non-segmented RNA viruses) [3–15]. accession numbers of the polymerase genes used in this
The RG technology has also revolutionized the influenza study are described in Table 2.
field, progressing influenza research by way of genetically
engineered recombinant influenza viruses. Reverse genetics Amplification of Megaprimer
as a tool has helped in studying the influenza host range A target amplicon (known as a megaprimer) with 5′ and 3′
[16, 17], transmission patterns [18] viral genome replication, overhangs complementary to the cloning site in pHW2000
pathogenicity and virulence [19–21]. This technique has plasmid which can facilitate the annealing of the megapri-
also been implemented to develop influenza vaccines [22, mer with the template plasmid was generated by PCR. The
23] or recombinant influenza viruses harbouring reporter PB1 megaprimer of A/Chicken/Vietnam/H7F-14-BN4–
genes for studying virus egress and dissemination [24]. 315/2014/H9N2 [Vietnam/2014], A/chicken/Bangladesh/
Despite the utility of RG systems, the cloning step 23527/2014/H9N2 [Bangladesh/2014], and A/chicken/
remains a limiting factor for the de-novo generation of vi- Jiangxi/02.05YGYXG023-P/2015/H5N6 [Jiangxi/2015] vi-
ruses. Gene cloning is a crucial step in RG technology and ruses and PB2 megaprimer of Vietnam/2014 virus were
has gained popularity in terms of usage but the technique amplified from the respective amplicons present in the
involves restriction digest [25] followed by ligation, which pMKT or pMK-RQ cloning vector (GeneArt® -Thermo
sometimes becomes difficult to perform. The primary rea- Fisher Scientific) using the primers mentioned in Table 1.
sons are presence of internal restriction enzyme sites (eg. The PB1 megaprimer was also amplified from the viral
for BsmBI, BsaI, AarI or BbsI) in the different gene seg- RNA of Vietnam/2014 virus. Briefly, the viral RNA was ex-
ments of field isolates of influenza virus. Further, the deg- tracted using QIAamp Viral RNA Mini Kit (Qiagen) and
radation of dNTPs in the ligation buffer or inefficient ligase reverse transcribed using Verso cDNA Synthesis Kit (Ther-
enzyme also result in failure during ligation. The RG plas- moFisher Scientific) and universal 12 primers (Uni 12) [31].
mids harbouring large inserts (> 2000 bp) of influenza virus The PB1 megaprimer was amplified using 20 nM of for-
gene segments have also been shown to be unstable after ward and reverse primers (Table 1) and Pfu Ultra II hotstart
transformation into E. coli cells [26, 27], which may be due PCR master mix (Agilent Technologies). The PCR amplifi-
to their toxicity to the bacterial host [28, 29]. This leads to cation steps include: initial denaturation at 95 °C for 3 min,
incorporation of bacterial sequences into the target insert. followed by 35 cycles of denaturation (95 °C for 30s),
As such, an alternative strategy for cloning is sought after. annealing (58 °C for 30 s) and extension (72 °C for 3 min)
Ideally it would bypass the restriction-ligation steps, in- followed by final extension (72 °C for 5 min). The amplified
crease the efficacy of recombinant plasmid formation and virus gene-specific amplicons (Megaprimers) were purified
reduce the chances of genetic recombination in the insert. using QIAquick gel extraction kit (Qiagen) and quantified
Taking these aims into consideration, we have developed a using nanodrop (ThermoFisher Scientific).
ligation and restriction enzyme independent (LREI) cloning
procedure for cloning influenza gene segments into the Cloning PCR, DpnI digestion and transformation
standard reverse genetics pHW2000 plasmid [30]. LREI Purified megaprimers (250 ng) were subjected to ther-
cloning increases the chances of recombinant plasmid for- mocycling using Pfu Ultra II hotstart PCR master mix
mation which if followed by growing bacteria at lower tem- (Agilent Technologies) using pHW2000 containing Poly-
peratures alleviates the problem of genetic recombination. merase Acidic (PA) gene from A/chicken/Bangladesh/
Our work would be particularly beneficial to researchers 23527/2014 (H9N2) [Bangladesh/2014] (100 ng) (Fig. 1).
who utilise the pHW2000 plasmid in RG workflows with The thermocycling conditions include: initial denatur-
influenza virus genes. ation at 95 °C for 4 min followed by 45 cycles of denatur-
ation (95 °C for 30 s), annealing (55 °C for 30 s) and
Materials and methods extension (72 °C for 6 min) followed by final extension at
Primer design 72 °C for 10 min. DpnI digestion of the parental template
Primers were designed in such a way that the 5′ end of was carried out by addition of 3 μl of DpnI to the re-
the forward primer contains 16 nucleotides complemen- action mix and incubated at 37 °C for 2 h. The reaction
tary to the one end of pHW2000 multiple cloning site was stopped by heating at 80 °C for 10 min. 15 μl of the
Bhat et al. Virology Journal (2020) 17:82 Page 3 of 9

Table 1 Primers used for amplification of PB2 and PB1 gene segments and subsequent cloning into pHW2000 vector
Gene Forward Primer (5′ to 3′) Reverse Primer (5′ to 3′) Expected Size (bp)
PB2 TCCGAAGTTGGGGGGGAGCGAAAGCAGGTC CCGCCGGGTTATTAGTAGAAACAAGGTCGTTT 2370
PB1 TCCGAAGTTGGGGGGGAGCGAAAGCAGGCAAAC CCGCCGGGTTATTAGTAGAAACAAGGCATTT 2370
The forward and reverse primers contain 16 and 13 nucleotides, respectively which are complementary to the pHW2000 (italics), followed by 12 and 13
nucleotides forming conserved UTR (bold) and 2–6 gene specific nucleotides (underlined). The nucleotide sequences (in italics) anneal to the pHW2000 and allow
directional cloning of desired gene segment into the pHW2000 vector

DpnI digested product was used for transformation of Ligation and restriction enzyme independent (LREI)
one shot DH5α cells (Invitrogen)/XL-Gold competent bac- cloning procedure using PA-pHW2000 as a bait plasmid
teria. The plates were incubated overnight at 32 °C. The improved the cloning efficiency
PB2 and PB1 positive colonies of Bangladesh/2014 and The ligation independent cloning PCR was initially per-
Vietnam/2014 H9N2 were screened by plasmid PCR of the formed by using megaprimers and empty pHW2000 or
extracted plasmid using Hoffmann primers [31]. The PB1 pHW2000 containing M gene as a bait plasmid. But this
positive colonies of Jiangxi/2015 were screened by colony didn’t yield any positive colonies after DpnI digestion
PCR using the primers H5N6 PB1F (GAAGTTGGGGGG and transformation. Since, the three polymerase genes
GAGCGAAAGCAGGC) and H5N6 PB1-R (CATCAC (PB2, PB1 and PA) contain same UTR sequences, thus,
ATCCTTGAGGAAATCTATTAG) followed by confirm- to improve the annealing efficiency of megaprimer with
ation by plasmid PCR using Hoffmann primers [31]. The the bait plasmid, pHW2000 containing PA gene was
desired recombinants were further confirmed by nucleotide taken as a bait plasmid (Fig. 1) and cloning PCR steps
sequencing using T7F and bGH R primers. were performed. Transformation followed by growth of
transformed bacteria at 37 °C resulted in colonies which
showed PB2 and PB1 specific bands after screening with
Virus rescue
PB2 and PB1 specific primers. However, nucleotide
A standard influenza virus reverse genetics protocol was
sequencing of the plasmids showed some foreign gene
followed to rescue influenza viruses [1]. Briefly, 1 μg of
insertions, which possibly were inserted by genetic
recombinant DNA plasmids carrying Vietnam/2014
recombination with the bacterial sequence.
virus cDNAs were mixed with Lipofectamine 2000 (Life
Technologies) and transfected into HEK-293 T cells. At
Growth of recombinant culture at 32 °C reduced bacterial
24 h post-transfection, the HEK-293 T cells were co-
recombination
cultured with MDCK cells in the presence of TPCK
To reduce the bacterial recombination between the de-
treated trypsin (Sigma). After 72 h post co-culture, pres-
sired plasmid and bacterial genome, temperature for
ence of rescued recombinant virus in culture superna-
growth of the recombinant culture was reduced to 32 °C
tants was confirmed by standard Haemagglutination
from 37 °C. Colony screening by plasmid PCR (Figs. 2, 3
Assay [32] and Plaque Assay [33].
and 4) showed PB2/PB1 specific colonies and further
nucleotide sequencing showed desired PB2 and PB1
Results inserts without any recombination.
PB2 and PB1 could not be cloned by conventional
restriction digestion and ligation LREI method saves time when cloning involves multiple
The PB2 and PB1 genes of H5N6 and H9N2 viruses gene segments
(Table 2) could not be cloned into pHW2000 by stand- The LREI cloning method is faster than the conventional
ard cloning procedures as mentioned by Hoffmann et al cloning methods due to bypassing the restriction diges-
[30]. Either there were no colonies, or the 20–40 tion of the insert amplicons and plasmids and the
colonies screened were all negative for the desired insert. ligation procedures.

Table 2 Accession number (retrieved from GISAID/NCBI) of Polymerase genes used in the present study
Gene Virus Subtype GISAID ID/ Blasts with E. coli type
NCBI Accession Number
PB2 A/chicken/Bangladesh/23527/2014 H9N2 KT362035 2012-EL-2448
PB1 A/chicken/Vietnam/H7F-14-BN4–315/2014 H9N2 EPI_ISL_327772 K12
A/chicken/Bangladesh/23527/2014 H9N2 KT362036 K12
A/chicken/Jiangxi/02.05YGYXG023-P/2015 H5N6 EPI_ISL_199401 K12
Nucleotide blast of PB2 and PB1 shows atleast 1% homology with bacterial genome
Bhat et al. Virology Journal (2020) 17:82 Page 4 of 9

Fig. 1 A schematic representation of the LREI cloning procedure. The technique involves designing primers that incorporate the gene specific
untranslated region (UTR) (yellow) and nucleotides homologous to the plasmid pHW2000 (red) multiple cloning site (MCS) to the polymerase
coding region resulting in formation of a megaprimer. The viral RNA extracted from the influenza virus (1) can be reverse transcribed by using
universal 12 primers (AGCAAAAGCAGG) (2), and the megaprimer can be amplified either from cDNA or any donor plasmid using the primers
mentioned in Table 1 (3). Denaturation of the megaprimer generates two primers having complementary ends to the pHW2000 MCS, which
when used with a bait plasmid facilitate annealing (4,5). Subsequent thermocycling steps result in extension of the annealed primers (5) thereby
synthesizing the non-methylated DNA insert along with the pHW2000 vector as a mixture. DpnI treatment results in digestion of parental
methylated DNA, (7) leaving the newly synthesized non-methylated DNA, which can be transformed into E. coli resulting in formation of the
desired recombinant plasmid (8)
Bhat et al. Virology Journal (2020) 17:82 Page 5 of 9

Fig. 2 The PB1 positive colonies of Bangladesh/2014 and Vietnam/2014 H9N2 virus were screened by plasmid PCR using the PB1 primers [31].
Out of 16 colonies screened for Bangladesh PB1 (lane 1–16), 3 were positive (13, 14, 15; indicated by arrows) while for 9 colonies screened for
Vietnam PB1 (lane 17–25), 4 were found to be positive (22, 23, 24, 25; indicated by arrows). The positive clones were further confirmed by
nucleotide sequencing using T7F and BGHR primers. (+ = positive control; M = DNA marker)

Fig. 3 The PB2 positive colonies of Bangladesh/2014 H9N2 virus were screened by plasmid PCR using the PB2 primers [31]. Out of 47 colonies
screened (lane 1–47) for Bangladesh/2014 PB2, 7 were positive in plasmid PCR (indicated by arrows). The positive clones were further confirmed
by nucleotide sequencing using T7F and BGHR primers. (+ = positive control; M = DNA marker)
Bhat et al. Virology Journal (2020) 17:82 Page 6 of 9

in generation of desired recombinant plasmid without

involving restriction digestion and DNA ligation. Fur-
thermore, the unique overhang present in the megapri-
mer results in directional cloning of the desired insert.
This was confirmed by Sanger sequencing of the cloned
plasmids. The LREI cloning strategy allows for the inte-
gration of any gene into any site of the vector, provided
the same strategy of megaprimer is followed.
The pHW2000 vector is a bidirectional plasmid that has
RNA PolI and PolII promoters for the generation of influ-
enza vRNA and mRNA. Thus, cloning a cDNA copy of all
the gene segments into pHW2000 plasmid and transfection
of recombinant plasmids into HEK-293 T cells results in de
Fig. 4 The PB1 colonies of Jiangxi/2015 were screened by colony novo generation of influenza virus [30]. Cloning remains a
PCR. A portion of 550 bp of PB1 was amplified using the primers critical factor for the rapid generation of influenza viruses
H5N6 PB1F (GAAGTTGGGGGGGAGCGAAAGCAGGC) and H5N6 PB1-R in vitro. As a proof of principle for establishment of the
(CATCACATCCTTGAGGAAATCTATTAG). Eight colonies were screened LREI cloning procedure, we sub-cloned the PB2 gene of
by colony PCR. The positive colonies in colony PCR (indicated by
Bangladesh/2014 H9N2 and PB1 genes of Vietnam/2014
arrows) were further confirmed by plasmid PCR and nucleotide
sequencing using T7F and BGHR primers (+ = positive control; H9N2, Bangladesh/2014 H9N2, and Jiangxi/2015 H5N6
M = DNA marker) from pMKT/pMK-RQ vector (GeneArt®) into the
pHW2000 vector. The technique involved choosing a bait
plasmid that can assist in formation of a desired recombin-
The cloned plasmids can efficiently be used to generate ant. In the first instance, using empty pHW2000 and
viruses de-novo pHW2000 vector containing Matrix (M) gene as bait plas-
To confirm the functionality of the cloned genes, the mids didn’t result in any colonies being formed, possibly
plasmids were checked for their ability to rescue influ- due to instability of the overloop (Fig. 1) [5]. We assumed
enza virus in-vitro. All the polymerase plasmids were the size difference between the desired insert and the insert
functional and reassortant viruses could be rescued by present in the bait plasmid could be a key to generate the
using the 8 plasmid reverse genetics system. desired recombinant plasmid. Since the conserved nucleo-
tides in the UTR of the Polymerase genes are similar, to
Discussion minimise the size of the overloop formed during the ther-
Here we developed a novel LREI directional cloning mocycling with the megaprimer and to maximize the possi-
technique which bypasses the restriction digestion step bility of generation of successful recombinant by cloning
and thus can be employed to increase the cloning effi- PCR, we used pHW2000 containing PA insert as a bait
ciency of influenza gene segments having internal re- plasmid (Fig. 1 ) [4].
striction sites into pHW2000 vector. The primers (Table While doing LREI cloning, we experienced bacterial re-
1) were designed to target conserved non-coding region combination in the desired gene, which normally occurs
(NCR) of influenza genes such that the approach can be as an outcome of increased metabolic burden on recom-
used for targeted cloning of any gene of influenza A binant bacteria, due to concatemer rich sequences in the
virus with the exception of few subtypes of Neuraminid- insert [34, 35] secondary or tertiary structures in the
ase (NA) due to three nucleotide difference at the 3′ and DNA, too low or too high copy number of the plasmid
5′ ends in their NCR [31]. This technique used PCR to [36], genotype of the competent cells [37, 38], length of
create a target amplicon known as a megaprimer with 5′ the cloned segment or temperature used to grow the cul-
and 3′ overhangs. These overhangs are complementary ture [39, 40]. Although the exact reason for experiencing
to the cloning site in pHW2000 plasmid and facilitate difficulties in cloning is difficult to pin point, there seemed
the annealing of the megaprimer with the template plas- to be no convincing role of DNA secondary structures or
mid. Thermocycling was conducted to anneal the mega- GC content (data not shown). However, nucleotide blast
primer with pHW2000, which results in the formation showed that PB1 gene segments had around 1% sequence
of an overloop (Fig. 1). However, to minimise the homology with the E. coli K-12 genome which is the pro-
chances of self-annealing of megaprimers and to in- genitor of most of the commercially available lab strains
crease the chances of annealing with the bait plasmid, a of E.coli. Based on the published reports, sequence hom-
higher concentration of the bait plasmid can be used. ology can contribute to homologous recombination lead-
DpnI digestion of the parental methylated DNA followed ing to deletion/insertions in target insert [41]. Thus, as an
by transformation of the nicked-circular plasmid results effort to reduce the metabolic burden on the transformed
Bhat et al. Virology Journal (2020) 17:82 Page 7 of 9

bacteria, all the incubation steps involving growth of re- in reverse genetics system. The technique has been used
combinant bacteria were performed at 32 °C instead of to clone the Neuraminidase (NA) gene of a field isolate of
37 °C. Nucleotide sequencing of all the plasmids for target H9N2 virus having internal restriction sites for BsaI [42]
gene inserts further confirmed the presence and sequence using pHW2000 containing M gene as a bait plasmid.
orientation of the desired gene and absence of transpos- Likewise, the bait plasmid containing M gene can be used
able elements. The colonies that carried PB2 and PB1 to clone Haemagglutinin (HA) and Nucleoprotein (NP)
gene segments were also found to be relatively smaller in genes into pHW2000. For cloning of smaller segments like
size, compared to other colonies which were negative by M and Non-Structural (NS) genes, empty pHW2000 vec-
PCR, suggesting that small colonies likely contain the plas- tor can be used as a bait plasmid. LREI cloning technique
mids that incorporate the correct length PB2 and PB1 is also quicker and takes less than 2 days for cloning and
gene insert in contrast to the larger size colonies which confirmation of the desired clone (Table 3). Furthermore,
generally contained empty plasmid or a plasmid with the cloned cDNAs could efficiently generate influenza vi-
shorter or truncated versions of the gene inserts [26]. This ruses de novo. Thus, our LREI technique is more robust
can potentially also be due to the metabolic burden on the and efficient for de-novo synthesis of influenza viruses and
recombinant bacteria, which could be associated with the complements the 8 plasmid reverse genetics system.
plasmid DNA replication and which eventually leads to re- Various strategies for cloning have been reported, which
duction in the growth rate of the recombinant bacterial include: TA cloning [43], GATEWAY recombinational
cells [39]. However, growing the recombinant bacteria at cloning [44], CloneEZ one step cloning [45], and cloning
32 °C doesn’t necessarily prevent the insertion of bacterial by overlap extension PCR [46]. Each method has its own
sequences into the cloned influenza gene. Although we limitations e.g. TA cloning using standard Taq DNA Poly-
did not notice any recombination in the polymerase genes merase may result in point mutations in the amplicon
grown at 32 °C in the present study, we have encountered during PCR amplification of the desired amplicon and fur-
the problem of genetic recombination while doing site- ther required specific sequences to create overhangs that
directed mutagenesis of smaller segments like HA and NS would facilitate cloning procedures. Another technique
even at 32 °C. This was countered by further reduction of called Gateway recombinational cloning requires DNA re-
temperatures to 30 °C or sometimes the recombinant cul- combination to transfer DNA between donor and destin-
tures were incubated at room temperature. However, this ation vectors, but this requires additional sequences for
reduces the bacterial growth in the recombinant culture recombination. CloneEZ kits use sticky ends in the vector
and can affect the plasmid yield. and insert for cloning but linearization of vector by
To confirm the efficacy of the proposed method, LREI restriction digestion is required.
cloning was also utilized to clone PB1 of Vietnam 2014 Like LREI, another approach involving the use of ccdB
H9N2 by using megaprimer amplified from viral RNA gene as a selection marker has also been used to insert
using RT-PCR employing PB1 specific primers (Table 1). the influenza PB2 and PB1 genes into the pHWSccdB
Our LREI cloning procedure can efficiently be employed vector [47]. Our approach neither requires any selection
to clone influenza gene segments from field isolates hav- marker nor is there any requirement for modification of
ing internal restriction sites for the standard enzymes used pHW2000 vector. Similarly, many novel approaches to

Table 3 Comparison of relative time taken by LREI cloning compared to conventional cloning
Steps in cloning procedure Time required during different cloning steps
Conventional cloning Ligation and Restriction Enzyme
Independent (LREI) Cloninga
a Viral RNA extraction and cDNA synthesis 2.5 h 2.5 h
b Generation of desired amplicon by thermocycling 3–5 h 3–5 h
c Agarose gel electrophoresis and gel extraction of desired 2h 2h
amplicon
d Restriction digestion of desired amplicon and Restriction 1 h – 16 h (depending upon the Not required
digestion of the cloning vector enzyme used)
e Purification and quantification of the digested amplicon and 1–2 h
the cloning vector
f Quick ligation or overnight ligation of the digested amplicon 1 h – 16 h (depending upon the
and cloning vector ligation kit)
g Transformation 1.5 h 1.5 h
h Screening of positive colonies 3–7 h 3–7 h
a
Our LREI cloning saves 2 days of time while cloning and is more efficient in cloning unstable polymerase genes into standard cloning vector
Bhat et al. Virology Journal (2020) 17:82 Page 8 of 9

molecular cloning including Homologous recombination Ethics approval and consent to participate
[48, 49], PLICing [50] and use of Zinc finger nucleases Not applicable.

[51] have been proposed in recent years that also don’t

Consent for publication
require restriction enzymes. Many researchers have re-
Not applicable.
ported similar strategies of DNA cloning by PCR which
include restriction site-free cloning [52], restriction free Competing interests
cloning [53], cloning by overlap extension PCR [46] and The authors declare that they have no competing interests. The funders had
MEGAWHOP cloning [54]. no role in the design of the study; in the writing of the manuscript, or in the
decision to publish the results.
Our LREI cloning technique is based on exponential
amplification of a megaprimer and the targeted vector, Received: 27 December 2019 Accepted: 17 June 2020
which results in a greater number of positive colonies
after transformation compared to the conventional clon-
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