There exist a lot of vectors for a wide range of experiments, and new modified plasmids appear every year. Most of them contain
different Multiple Cloning Regions (MCR)s depending on specificity of the experiments. The main problem of such the trial-and-error
method is that a lot of MCRs contain a limited number of Restriction Sites (RS), which often makes the process of subcloning arduous.
Here, we elaborated design of the MCR for a GeneClip vector that can be used both for cloning PCR products and transferring the
insert from one plasmid to the others. So, everybody can now construct MCRs for their own vectors using this manual.
PCR Products Cloning, T-vector, subcloning, Multiple Cloning Region (MCR), Restriction Sites (RS)
The polymerase chain reaction (PCR) method enables one to
amplify a single DNA (or RNA) segment across several orders of
magnitude, generating the multitude of copies of a particular DNA
sequence. Since polymerase chain reaction was invented new
modified PCR applications appeared to perform a wide range of
genetic manipulations[2, 3, 4].
Different DNA polymerases can be used to optimize DNA
amplification depending on the aim of experiment. Thermostable
DNA polymerases such as Taq, Tth, Tfl are most often used to
amplify the DNA fragments. These polymerases don’t have
proofreading activity but can add a single base extention of
deoxyadenosine to the 3´-ends of amplified products. 3’-Ts
overhanging on DNA duplexes complementary to adenines of the
PCR products allow to clone them directly into linearized T-vectors
avoiding restriction and ligation[5, 6]. Blunt-ended products
derived from PCR with proofreading Pfu and Tli polymerases can
be modified with 3’-overhangs by incubating the PCR fragment
with dATP and a nonproofreading DNA polymerase[7, 8, 9].
There is a variety of T-vector systems designed for efficient
cloning of PCR products. T-ends of such T-vectors are surrounded
by MCRs containing RE sites that can be used for the inserts
transferring. However sets of RS are often limited and it is difficult
to select a suitable site for subcloning the insert from a subsidiary
vector to the destination one. Though subcloning is a basic
molecular biology procedure, it claims a lot of time, and is not
very handy when the experiment requires a number of repeats and
variants or confirmations with a different object or insert. We tried
to overcome these difficulties through design of universal MCR
that can be used both for cloning PCR products and transferring
the insert from one plasmid to the others.
Materials and Method:
Construction of Multi Cloning Region for the GeneClip vector:
The main idea of our experiment was preparing of double-stranded
oligonucleotide as a polylinker with two XcmI recognition
site variants for GeneClip vector. XcmI recognition sites were
placed in such a way that a digestion of the modified vector with
endonuclease XcmI could give rise to 3’-Ts overhanging on DNA
duplexes complementary to adenines of the PCR products. It makes
possible to clone PCR products directly into linearized T-vector.
RE sites for the MCR design were determined by the trial-anderror
method with the help of SnapGene Viewer 2, 5 software
, and online tool Enzyme Finder NEB. Oligonucleotides
mcsT1and mcsT2 (reference sequences are listed in the
supplementary material) were synthesized in vitro by Syntol
Company, Russia. Equimolar amounts of oligonucleotides were
annealed by incubating in restriction buffer (BSA-free) for 5 min
at 950С, for 15 min at 650С in water bath, and then cooled to room
temperature. The synthetic MCR obtained was stored at -200С no
longer than for a month.
For ligation of MCR with the GeneClip vector T4 DNA ligase with
accompanying buffer was used. 50ng of the dissolved GeneClip
vector, 5ng of annealed oligonucleotides, 25µl of 2xRapid ligation
buffer, 1µl of T4 DNA ligase (3 units) and sterile water to final
volume 50µl were mixed in a sterile micro centrifuge tube. Reaction
mix was incubated in thermo cycler for 1 hour at +210С and for 3
hours at +160С, then purified with the help of Wizard SV columns,
Promega, according to original protocol, and concentrated in 20µl
of sterile water.
Transformation of competent cells with purified ligation mix
was carried out as following. 50µl of defrosted JM109 cells were
incubated with 5µl of ligation mix in sterile 2ml tube for 20min
on ice, and after 45sec of heat shock at +420C were placed back
on ice for 5 min. A sample was supplemented with 250ml of SOC
media, incubated in a thermostatic shaker at +370C, 225 rpm for
an hour. Then cells were spread over LB agar plates and incubated
at 370C overnight.
For screening analysis 5 random colonies from plates were
incubated in sterile 2ml tubes with 1ml of LB media with
ampicillin in a thermostatic shaker at +370C, 225 rpm overnight.
200µl from each sample were then centrifuged at 5000g for 5
min. After supernatant removing, the pellets were suspended
in 20µl milliQ water and incubated in boiling water bath for 10
min. After centrifugation plasmid eluates were analyzed on PCR
amplification with gC(f) and gC(r) primers (reference sequences
are listed in supplementary material). Samples with positive
signals were isolated from 100 ml LB cell culture with the use of
PureYield Plasmid Midi Prep System, Promega, and analyzed then
by automatic sequencing. The probes were stored at -700C with
glycerol added in equal proportions.
GeneClip-T-vector preparing and PCR products cloning:
Plasmid DNA isolation was carried out with PureYield Plasmid
MidiPrep System according to an original protocol (Promega).
5µg of a positive sample of plasmid DNA were digested in
50µl volume with 10 units of endonuclease XcmI in conditions
of original restriction buffer NEB for 3 hours at +370С. After
GeneClip-T-vector digestion with XcmI reaction mixture was
exposed to about 30 min gel-electrophoresis in 0.8% agarose,
linearized GeneClip-T-vector was isolated from gel, purified with
Wizard SV columns, Promega, and dissolved in sterile water to
final concentration of 50ng/µl.
About 200bp of human pre-promoter rIGS region (3S fragment)
were amplified with primers N (f) and N (r). We used PCR gene
Pak Core System, Isogene, Russia with lyophilized components
including Taq DNA-polymerase. PCR product was purified
on Wizard SV columns and cloned into GeneClip-T-vector, as
Results and Discussion:
For classification of a new putative microRNA in the human
rDNA pre-promoter region a destination pGeneClip™ Vector was
selected. The GeneClipU1 Hairpin Cloning Systems (Promega)
contain linearized plasmids designed for cloning of human target
sequences to express short hairpin RNAs (shRNAs) in human
To place DNA fragment coding hairpin shRNA into the
pGeneClip™ Vector, two short oligonucleotides should be
annealed to form a DNA insert containing the sequence of interest.
After annealing, the oligonucleotides form definite overhangs that
are compatible with the pGeneClip™ Vector ends and facilitate
sticky-end ligation (Figure 1). Design of the insert for GeneClip is
limited by the length of the oligonucleotides that can be synthesized
in vitro, so fragments more than 200 bp can’t be inserted in
For cloning of PCR products into MCR it should contain a couple
of RE sites which are able to generate T-overhangs at 3’-ends.
Although such enzymes are very few in number, there exist some
enzymes with single-base 3´ extension
(XcmI enzyme as an example where N can imply any nucleotide):
With the help of such enzymes a T-vector can be derived from
any given plasmid by an insertion of the two suitable RE sites
into its MCR where 3’-ends of the both complementary chains are
represented by thymine. One should take into account that it is
necessary to place some nucleotides to separate the two RE sites
that produce T-overhangs for more effective enzyme action. This
separating site can be presented by another RE site as well. It is
crucial to choose enzymes that do not cut the destination vector.
The easiest way to do so is to compare the enzyme of interest with
the list of noncutters for the vector.
We managed to design the MCR for the chosen GeneClip vector
that makes it serve either as a T-vector for PCR products cloning or
as an intermediate vector for subcloning inserts into other vectors
After performing the cloning of DNA duplex into GeneClip vector
we checked if the MCR works properly by restriction of present
sites. Fragments of expected numbers and lengths were shown on
gel-electrophoresis (Figure 3A). Additionally the compliance of
designed MCR with this region in the recombinant plasmids was
verified by sequencing.
After linearizing the vector with XcmI enzyme we inserted into it a
PCR product of 209bp length from the human rDNA pre-promoter
region (3S fragment). Its presence was tested using primers
from the vector surrounding the insert; its orientation was shown
with the help of one primer from the vector and the other one from
the insert (Figure 4). Additional sequencing of recombinant
plasmids obtained showed the compliance with the expected
A Gel-electrophoresis of restricted fragments for 9 RE sites present in MCR. As it can be observed from the photo, RE sites
EcoNI, PacI, AscI, PmlI, SbfI are unique for the obtained GeneClip-T-Vector and can be used for subcloning if needed. Despite the fact
that XcmI is a double-cutter for the plasmid, the only product of 5kb is observed because the second one is too small. B Multiple Cloning
Region of the plasmid obtained. Primers surrounding the insert that were used for screening analysis are shown with arrows.
Gel-electrophoresis of PCR products of the GeneClipT+3S construction with specific primers gC(f) and N(r)
1, 2, 5 contain 3S insertions in direct orientation. Insertions in the remaining probes have reverse orientation or are absent at all.
The major practical significance of the method proposed is
enlarging of the potential of using common vectors and specific
vectors as well. Transforming a vector of interest into a T-vector
saves hands-on time and helps avoiding problems caused by
mistakes during intermediate stages of subcloning. Personal MCR
design furthers the process of individualization and allows us to
use available enzymes, vectors and other reagents.
One more advantage of using synthetic MCR is that it allows
having vectors and MCR itself in abundance. Since the plasmid
with such an insert is not linearized it can multiply in a cell and be
easily isolated when needed.
This study was partly supported by RFBR (project no 16-04-
00178), the RAS Program “Molecular and Cell Biology”, and the
RF President’s Program (no 86-12.2016.4).
Conflict of Interests:
The authors have declared that no conflict of interest exists.
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