Molecular cloning Techniques, process,types of cloning, primer designing for PCR, applications in drug discovery

1. ROLE OF
CLONING
METHODS
IN DRUG
DISCOVERY
PRESENTED BY
LIJO MANI
PC/2022/207
Under the guidance of
Dr. Santosh Kumar
Guru

2. INTRODUCTION
MOLECULAR CLONING
PLASMID
CLONING PROCESS
CLONING METHODS
PRIMER DESIGNING
COMPARISON OF DIFFERENT CLONING METHODS
APPLICATIONS
CONCLUSION
REFERENCES
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3. Clone: A group of genetically identical organisms/group of cells derived
from a single parent cell.
Cloning: Process of creating genetically identical copies of a biological
matter; includes genes, cells, tissues or entire organisms.
 Natural cloning
 Artificial cloning are of 3 types; Reproductive, Therapeutic &Molecular.
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4. Two main components of a Molecular cloning reaction are;
 DNA fragment(Insert/GOI)to be replicated
 Vector-A DNA molecule which can carry a DNA insert to generate
recombinant DNA & replicate in a particular host
Process by which recombinant DNA molecules are produced & transformed into a host organism,
where they are replicated.
Cloning vectors
• Plasmid
• Cosmid
• Phagemid
• Bacteriophage
• Artificial
chromosome
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Sambrook et al.,2012

5. A small, extrachromosomal circular DNA which can replicate independently and are physically
separated from chromosomal DNA in a bacteria.
 It can hold up to 20kb of foreign DNA.
Common structural components(plasmid backbone) are;
 Origin of replication
 MCS
 Selectable marker
 Promoter
 Protein tag
 Poly-adenylation signal
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6. Selection of cloning vector
Selection of host organism
Preparation of vector &
host(Restriction digestion/PCR)
Generation of recombinant DNA
using DNA ligase.
Introduction of rDNA into the
host(Transformation)
Selection of clones of organisms
containing rDNA
Screening of clones for desired
gene inserts and biological
properties
Expansion & isolation of rDNA
Plasmids
• pBR322
• pUC19
• pGEX
• pMAL
Ecoli Strains
• DH5α
• Stbl2
• JM110
• XL10 Gold
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Sambrook et al.,2012

7. Transformation- Process by which an
organism acquires exogenous DNA.
 It can be natural or artificial
Competent cell
 Chemical competence
 Electroporation
Selection
 Blue white screening
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8. Screening of clones for desired gene insert
 Restriction digestion screening
 Colony PCR
 Sanger sequencing
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9.  PCR/TA cloning- PCR amplified inserts
contain an adenine residue at the 3ʹ end
of DNA fragments(A-tailed ends)
 No need of RE and PCR products can be
used directly without modification.
 TOPO-using DNA Topoisomerase 1
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Bertero A et al.,2022

10. Primer is a short synthetic oligonucleotide designed to have a sequence which is the reverse
compliment of a region of template/target DNA
 Determine the target DNA sequence
 Choose the PCR cloning strategy
 Identify the primer binding sites
 Primer should be around 18-30bp & Tm b/w 55-65℃
 Avoid primer dimer formation, 40-60% GC content
 The Tm of two primers should be within 5℃ of each other
 Avoid secondary structures such as hairpins/loops
 Check for specificity
 Add restriction sites
 Validate the primers
PCR primer
• Leader sequence
• Restriction site
• Hybridization
sequence
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11. 11
Celie et al., 2016. Recombinant cloning
strategies for protein expression

12. Cloning
method
Sequence
dependency
Throughput
Assembly of
multiple
fragments
Directional
cloning
Need of
dedicated
vectors
Examples of
commercial
products
Traditional
(RE-based)
Yes(RES)
Low to mild(can
be increased by
using ligation
adapters)
Difficult for more
than 2 fragments
Possible No -
PCR Cloning No High
Challenging
(requires special
modification)
Difficult Yes TOPO® TA
Ligation
independent
Cloning
Limited
(vector)
Low Yes Yes No In-fusion®
Seamless
Cloning
No Low Yes Yes No
Gibson
assembly
GeneArt®
Recombinatori
al Cloning
No High Challenging Yes Yes
Gateway®
Echo
cloning™
Creator™
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Bertero A et al.,2022

13. 13
Method Gibson TA Yeast-mediated
Ligation
independent
Recombinatorial
Advantages
One-step cloning
of up to 6 insert
fragments.
High success rate
No RE digestion
required
Inexpensive.
Large-sized final
construct can be
cloned
Neither RE
digestion nor
ligation is
required.
Cheap & fast
Single step of
homologous
recombination
Disadvantages
Single-stranded
overhangs might
form stable
secondary
structures.
Inserts <200bp
tend not to work
Lacks
directionality
Long growth
time.
Successful
transformation
and isolation of
DNA is difficult
Stable secondary
structures
Specific
recombination-
efficient vectors
are needed.
Expensive

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• Development of recombinant
vaccines
• Production of therapeutic protein
• Site directed mutagenesis
• Creation of animal models for drug
testing
• Gene therapy
• HTS
• Transgenic mice
• Cell line creation
Yadav et al., 2020

15.  Cloning methods have revolutionized many fields of science, including drug discovery. They
are essential tools that enable researchers to isolate, manipulate and study genes and their
corresponding proteins.
 Cloning plays a significant role in drug discovery by allowing for the production of therapeutic
proteins, the creation of animal models for drug testing, and the development of vaccines.
 They have also allowed for the identification and development of new drug targets, high-
throughput screening and in gene therapy.
 These methods have significantly contributed to the development of new drugs and therapies
for various diseases and have the potential to continue to do so in the future.
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16.  https://www.addgene.org/protocols/pcr-cloning/
 Green MR,Sambrook J. Molecular Cloning: A Laboratory Manual.4th edition.Cold spring Harbor Laboratory Press;2012.
 https://goldbio.com/articles/article/Molecular-Cloning-Detailed-intro
 Kouprina, N., Larionov, V. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic
biology. Chromosoma 125, 621–632 (2016).
 Sigma Aldrich Sanger sequencing https://www.sigmaaldrich.com/IN/en/technical-documents/protocol/genomics/sequencing/sanger-sequencing
 New England Biolabs Cloning and Synthetic Biology. www.neb.org
 Bertero A, Brown S,Vallier L et al. 2022.Author Manuscript
 GenScript Molecular cloning Handbook www.genscript.com
 J.S.Reece-Hoyes and A.J.M.Walhout et al.2023. Gateway Recombinational cloning. www.cshprotocols.org
 Li,M.Z. & Elledge S.J. 2012. SLIC: A method for sequence and logation independent cloning. In.J.peccoud (Ed), Gene synthesis: Methods and
protocols 51-59
 Celie et al.2016.Recombinant cloning strategies for protein expression;Current opinion in structural biology.38:145-154.
 https://www.sigmaaldrich.com/IN/en/technical-documents/technical-article/genomics/cloning-and-expression/blue-white-screening
 https://blog.addgene.org/plasmids-101-topo-cloning
 https://www.creative-biolabs.com/blog/index.php/recombinant-antibody/
 Yadav T, Srivastava N, Mishra G, Dhama K, Kumar S, Puri B, Saxena SK. Recombinant vaccines for COVID-19. Hum Vaccin Immunother. 2020
Dec 1;16(12):2905-2912.
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