Gene Cloning has brought a revolutionary change in the fields of Genetic Engineering and Biotechnology. Application of this technique has become an increasingly important tool in molecular research due to its simplicity, cost effectiveness, rapidity, and reliability.

1. Presented By-
Md. Imran Hossain

2. Contents
• Introduction
• Steps of Gene Cloning
• Choice of host organism and cloning vector
• Preparation of vector DNA
• Creation of recombinant DNA
• Introduction of recombinant DNA into host organism
• Selection of organisms containing recombinant DNA
• Applications
• Conclusion
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3. Introduction
• Gene Cloning is a molecular biological technique wherein exact copies of
clones of a particular gene or DNA sequence are produced using the
principles of genetic engineering.
• What is the purpose of gene cloning?
o Gene Cloning is used to create multiple copies of a desirable gene for
various purposes, such as sequencing, mutagenesis, genotyping etc.
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4. Steps of Gene Cloning
Choice of host organism and cloning vector
Preparation of vector DNA
Preparation of DNA to be cloned
Creation of recombinant DNA
Introduction of recombinant DNA into host organism
Selection of organisms containing recombinant DNA
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Figure: Gene Cloning.

6. Choice of host organism and cloning vector
• A cloning vector is a small piece of DNA in which a foreign DNA fragment can
be inserted for cloning purposes.
• E. coli as a cloning host and plasmid as a vectors are in common use because
they are technically sophisticated, versatile, widely available, and offer rapid
growth with minimal equipment.
• Desirable gene from the host organism is selected for cloning.
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7. Preparation of vector DNA
• The cloning vector is treated with a restriction endonuclease to cleave the
Vector DNA at the site where foreign DNA will be inserted.
• The restriction enzyme is chosen to generate a configuration at the cleavage
site that is compatible with the ends of the foreign DNA.
• This is done by cleaving the vector DNA and foreign DNA with the same
restriction enzyme, for example EcoRI.
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8. Preparation of DNA to be cloned
• For cloning of genomic DNA, the DNA to be cloned is extracted from the
organism of interest.
• The DNA is then purified using simple methods to remove contaminating
proteins.
• The purified DNA is then treated with a restriction enzyme to generate
fragments with ends capable of being linked to those of the vector.
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9. Creation of recombinant DNA with DNA ligase
• DNA prepared from the vector and foreign source are simply mixed together
at appropriate concentrations and exposed to an enzyme called DNA ligase
that covalently links the ends together.
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10. Introduction of recombinant DNA into host organism
• The DNA mixture, previously manipulated in vitro, is moved back into a living
cell, referred to as the host organism.
• This recombinant DNA can be transfer through-
o Transformation
o Transduction
o Heat shock
o Electroporation
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11. Selection of organisms containing vector sequences
• When bacterial cells uptake recombinant DNA, the selectable marker usually a
gene that confers resistance to an antibiotic such as ampicillin.
• Cells harboring the plasmid will survive when exposed to that antibiotic, while
those that have failed to take up plasmid sequences will die.
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12. Applications
• Genome organization and gene expression
• Genomic Library
• Production of recombinant proteins
• Transgenic organisms
• Gene therapy
• Growth hormone and Insulin production
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13. Conclusion
Gene Cloning has brought a revolutionary change in the fields of Genetic
Engineering and Biotechnology. Application of this technique has become an
increasingly important tool in molecular research due to its simplicity, cost
effectiveness, rapidity, and reliability.
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14. Thank You
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