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Molecular cloning
Chapter · January 2021
DOI: 10.1016/B978-0-12-821471-8.00011-8
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Praveen Rai Hemant Arya
National Institute of Plant Genome Research Ruhr-Universität Bochum
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Chapter 11
Molecular cloning
Praveen Rai and Hemant Arya
Department of Biotechnology, Central University of Rajasthan, Bandarsindri, Rajasthan, India
1. Overview
1.1 Molecular cloning
Molecular cloning is a powerful experimental technique to achieve multiple
copies of the desired gene which could be further used for various experiments.
To produce a recombinant DNA molecule, a DNA fragment gene to be cloned
is inserted into a circular DNA molecule known as a vector. The vector takes the
gene into a host cell, which is generally a bacterium, even though other living
cells like yeast cell and mammalian cell, may also be used [1, 2].
1.2 Component required for cloning
1.2.1 Insertion of the desired gene
Insert from a plasmid source
Digest the plasmid with a suitable restriction enzyme(s) to generate a DNA
fragment/gene that can be used to clone directly into the vector. For directional
cloning, two restriction enzymes needs to be used to produces the compatible
end. Run the digested product onto agarose gel and cut the appropriate band.
Further, elute the insert by using the gel elution spin column.
Insert from a PCR product
Gene of interest (Insert) can also be amplified using genomic DNA/cDNA in
a PCR machine. The specific primers will be designed to amplify the gene out
the complete genomic DNA. You may also add the desired restriction enzyme
sites in the primers to be inserted at N/C-terminal of the gene. The amplified
DNA fragment/gene having an appropriate restriction site will be run on the gel
to confirm the size of the product. The PCR product will be purified using spin-
column or run it into agarose gel and cut down the appropriate band and cleanup
the band with the gel elution spin column.
The Design & Development of Novel Drugs and Vaccines. https://doi.org/10.1016/B978-0-12-821471-8.00011-8
© 2021 Elsevier Inc. All rights reserved. 135
136 PART | C In vitro study
1.2.2 Vector
Cloning vector
Vectors are circular, double stranded, self-replicating plasmid made up of DNA.
Cloning vectors are generally used to insert foreign gene into the plasmid to
enhance the copy number of the desired gene. Examples are plasmids, bacterio-
phage, cosmid, bacterial artificial chromosome (BAC), yeast artificial chromo-
somes (YAC), and human artificial chromosome (HAC) [3].
Expression vector
Expression vectors are derived from the plasmid and it has a specific site for
promotor and operator to facilitate the binding and regulation of RNA poly-
merase involves in transcription and followed by translation. Therefore, these
vectors are used for the expression of the desired gene. Expression vectors
are selected on the basis of specific host system. For example, pET series,
pGEX series, etc. for bacteria-based expression system; pPICZA, pGAP, etc.,
for yeast expression, pcDNA, p3xFLAG-CMV, pCMV-3Tag, etc. for mam-
malian cell-based expression system, and pFastBac for insect cell-based ex-
pression, etc.
1.3 Types of cloning
There are many types of the cloning methods available to clone the gene based
on the reaction condition. Here are examples of some of those methods:
1. Restriction digestion based cloning
2. Gateway recombination cloning
3. TA cloning
4. Isothermal assembly reaction (Gibson assembly)
5. Type IIS assembly (golden gate/MoClo)
6. Ligation independent cloning
7. Yeast-mediated cloning and oligonucleotide stitching/yeast-mediated
ligation protocol
2. PCR (polymerase chain reaction)
PCR works on DNA polymerase’s ability to synthesize new DNA strand com-
plementary to the given template strand. Since DNA polymerase can only at-
tach a nucleotide to a pre-existing 3′-OH group, it includes a primer to which
the first nucleotide can be attached. This necessity actually defines a specific
region of the template sequence to be amplified. The particular sequence ac-
cumulated in billions of copies (amplicons) at the end of the PCR reaction
[4, 5] (Fig. 11.1).
Molecular cloning Chapter | 11 137
DNA Primer Nucleotides
Template 5’ 3’
3’ 5’
Denaturation
5’ 3’ 5’ 3’
Annealing
5’ 3’ 5’ 3’
Extension
3’ 5’ 3’ 5’
5’ 3’ 5’ 3’
FIG. 11.1 Overview of PCR amplification.
Requirements
1. DNA template
2. dNTP’s
3. 10 × buffer
4. Taq DNA polymerase
5. Forward and reverse primer
6. Nuclease free water
7. PCR tube
8. Equipment
9. Thermocycler
10. Micro pipet
Reaction mixture
TABLE 11.1 Standard polymerase reaction protocol.
For 50 μL of the
S. no. Reagent Final concentration reaction mixture
1 Nuclease free water – Optimizible
2 10 × Taq buffer 1× 5 μL
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