This Power point presentation describes various cloning strategies especially isolation of desired DNA/Gene to be cloned. It describes isolation of DNA/gene to be inserted in vector in 5 different situations.

1. Various Strategies for Molecular
Cloning
Gopal Jee Gopal, PhD(JNU)
Assistant Professor
C.G.Bhakta Institute of Biotechnology
Uka Tarsadia University, Bardoli

2. Definition
Cloning: Production/Copying of identical things
Molecular Cloning: Copying of DNA/gene

3. Why we need multiple copy of DNA?
Purpose of Molecular cloning?
Production of high amount of DNA
required for gene manipulation.
Production of recombinant protein for
several purpose.
Recombinant DNA technology is a fuel for
Biotechnology.

4. Application of Gene manipulation on
the practice of medicine
Principle of gene manipulation,7th Edition.

5. Agriculture
Pest resistant plant: BT Cotton, BT brinjal
High Mineral and Vitamin containing plant: Golden rice having high
carotene(precursor of Vit A) .
Drought resistant plant:
Herbicide resistant plant: by increasing no of copies of gene or
modifying binding site.
Virus resistant plant: Incorporation of gene which code virus coat
protein.( cross protection mechanism)
High quality and durable plant product: Flavor savr potato (delayed
ripening of fruit using antisense RNA technology.
Salt resistant plant:

6. Cloning is required for genetic
Engineering

7. What are the strategies to get
multiple copy of same DNA?
We can produce identical copy of given DNA Applying Recombinant DNA Technology(RDT)
in very less cost provided we must have our desired DNA inserted into a Vector(Molecular
cloning).
Steps of Molecular Cloning
Isolation of Desired DNA/gene
Joining with vector
Introduction recombinant vector into host cell
Screening of Positive Clone

8. Isolation of desired DNA to be cloned
Situation 1: We have to clone an H.Pylori gene coding for Cytotoxin(CagA) and we have also
H.pylori strain. Gene sequence of CagA is known. What are the steps of cloning/how can we
isolate the gene of interest, CagA to be cloned?
Situation 2: We want to produce Human protein A in E.coli. We know the
sequence of gene. How can we isolate our gene for cloning so that we can express cloned gene
in E.coli and produce recombinant human protein in E.coli?
Situation 3: We have to clone a gene ( encode Thermo polymerase Enzyme) to get its
recombinant protein. We cannot culture that organism but other scientist have reported and
deposited sequence of the gene in database. How can we clone this gene and produce
recombinant thermo polymerase protein?
Situation 4: We want to search the gene which code for beta galactosidase enzyme
in Bacillus. We know the bio assay of beta galactosidase enzyme. How can we proceed
to find out the corresponding gene ?( No information about gene sequence but we have
Bacillus bacteria).
Situation 5: We know the amino acid sequence of protein X of Yeast.(Through Purification of
endogenous protein followed by aminoacid sequencing). Can we find out corresponding gene?
How?

9. Situation#1
PCR based cloning: Specific gene will be amplified using
specific primer pair. PCR Amplified DNA/Gene will be
digested and ligated into pre-digested vector. Since
sequence of gene is known, Primer can be designed and
synthesized.

10. Hint for Situation:2
Eukaryotic genes are interrupted by introns. Splicing is needed before mRNA used for
translation. There is no system of splicing available in E.coli so Eukaryotic gene will not
express into protein. Therefore cDNA should be cloned not gene if purpose is to
produce recombinant protein in E.coli.

11. Situation #3
Direct Synthesis of Gene( Chemical synthesis).
We cannot amplify gene using PCR because template DNA ( Genomic DNA)
is not available.
Since gene sequence is known, Direct chemical synthesis of gene is possible.

12. Situation: 4
Genomic Library
cDNA Library
Library can be screened based on Bioassay
of protein produced by gene cloned in the vector

13. 5’-ATGCCTAGGTACCTATGA-3’
3’-TACGGATCCATGGATACT-5’
5’-AUG CCU AGG UAC CUA UGA-3’
N-MET-PRO-ARG-TYR-LEU-C
1 4 4 2 6
DNA (gene)
Transcription
Translation
mRNA
Protein
Hint for Situation: 5
•Its easy to determine protein sequence if gene sequence is known but determination
of gene sequence(original) from protein sequence is not possible because of
redundancy/degeneracy of genetic code).

14. Isolation of Gene/DNA to be cloned
1. PCR of
gene
2.cDNA
synthesis
4. Mechanical shearing
or enzymatic cleavage
3. Direct
synthesis
5. Degenerate PCR/
Redundant PCR

15. 2.Joining of DNA/Gene to Vector
1. Ligation with cohesive termini:
2. Homo polymer tailing: Blunt ended gene/vector can be made cohesive
ended using terminal transferase enzyme and specific nucleotide.
3. Ligation using Linker/adaptor: Blunt ended DNA can be made
cohesive ended by addition of specific short oligonucleotide having RE
recognition sequence/overhang.
4. Blunt end ligation:
5. TA cloning: Pre-digested vector ( TA Cloning kit)having ‘T’ overhang is
ligated with Taq amplified PCR product having single ‘A’ nucleotide overhang
using good quality of ligase enzyme supplied with kit.

16. Introduction of DNA/rPlasmid into
desired host.
 Transformation/Transfection
Transduction
Conjugation
Using Sphaeroplast method
By electroporation
Liposome method
Agarobacterium mediated transfer
Gene gun method.

17. Screening of colony which harbours
Positive clone( desired recombinant
Plasmid)
1. Blue/white Screening: (Only to distinguish self ligated plasmid and
recombinant plasmid)
2. Replica plating: (Only to distinguish self- ligated vector and
recombinant recombinant vector)
3. Double digestion: (Checking whether size of fall out is same to size
of inserted gene or not after double digestion of recombinant vector
isolated from transformed colony)
4. PCR/Colony PCR:

18. Screening.....
5.Hybridization( Colony or plaque hybridization):
6. Immunochemical method:
7. Protein/Protein Interaction:
8.Protein/ligand interaction:
9.Functional complementation:
10. Gain of function.

19. Summary of Gene Cloning
Fig: Adopted from Principle of Gene manipulation, 6th edition.

20. Thank You
For
Attention
Send your feedback at
gopaljigopal@rediffmail.com
Gopal.jee@utu.ac.in
or at 9558880617, 8160245501