Recombinant �DNA �Technology

Molecular Tools of r-DNA Technology -

1. Restriction EndoNuclease –

DNA cutting enzyme

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2. DNA Ligase –

DNA Sealing enzyme

What is �Restriction EndoNuclease?

• The bacterial enzymes that can cut/ Split DNA at specific sites.

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• Restriction EndoNuclease was first discovered in E. coli, restricting the replication of Bacteriophage, by cutting the viral DNA.

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• They are known as restriction enzymes or restriction EndoNuclease or Genetic Scissor.

Nomenclature -

• The first letter of the enzyme indicate the genus name, followed by the first two letter of the species, then comes the strain of the organism and finally roman numerical indicating the order of discovery.

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• E.g. EcoRI is from Escherichia (E) coli (co) Strain RY-13 (R) and first EndoNuclease (I)

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• Hind III Haemophilus (H) influenzae (in) Strain Rd (d) and third endonuclease (III)

Recognition Sequences -

• It is the site where the DNA is cut by restriction endonuclease.

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• Restriction endonuclease can specifically recognize DNA with particular sequence of four to eight nucleotides and cleave.

Cleavage Pattern -

What is DNA Ligase?

• DNA Ligase actively participates in cellular DNA repair process.

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• DNA Ligase joins (Seals) the DNA fragment by forming a

phosphor di ester bond

What are Host Cells?

• Factories of cloning

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• The living system or cells in which the carrier of recombinant DNA molecule or vector can be propagated.

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• Prokaryote (Bacteria) and Eukaryote (Fungi, animal and Plants)

What is Vector?

• Cloning Vehicle

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• DNA molecule which can carry a foreign DNA fragment

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• They are self replicating

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• E.g. Plasmids, Bacteriophage, Cosmids and artificial chromosome vectors.

What is Plasmid?

• Extra chromosomal double stranded circular self replicating DNA molecules.

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• All the bacteria have plasmids.

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• The size of plasmid vary form 1 – 500 kbp.

What is Transformation?

Recombinant DNA (rDNA) Technology

• Recombinant DNA technology refers to the joining together of DNA molecules from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry.
• Recombinant DNA (rDNA), on the other hand is the general name for a piece of DNA that has been created by the combination of at least two strands.
• They are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in the genome.

Brief History

• In 1968, Swiss microbiologist Werner Arber discovered restriction enzymes.
• American microbiologist Hamilton O. Smith subsequently identified type II restriction enzymes.
• Unlike type I restriction enzymes, which cut DNA at random sites, type II restriction enzymes cleave DNA at specific sites; hence, type II enzymes became important tools in genetic engineering.
• Daniel Nathans, 1970-71, demonstrated that type II enzymes could be usedful in genetic engineering.
• Paul Berg, 1970-71, developed methods for splitting DNA molecules at selected sites and attaching segments of the molecule to the DNA of a virus or plasmid, which could then enter bacterial or animal cells.

Recombinant DNA Technology

Pieces of DNA, such as human DNA, can be engineered in a fashion that allows them to be copied, or replicated, in bacteria or yeast. This involves attaching appropriate elements to a piece of DNA and then transferring into a bacterial or yeast cell, with those elements then instructing the bacterial or yeast cell to copy this DNA along with its own. This process is known as DNA cloning, with the resulting cloned DNA often referred to as recombinant DNA.

Principle of Recombinant DNA Technology

• Insertion of the selected DNA into a Cloning vector

Principle of Recombinant DNA Technology

• Introduction of Recombinant vector into host cells

Selection of recombinant cells

• Multiplication and selection of clone and Expression of the gene to produce the desired product

To be useful, the recombinant molecule must be replicated many times to provide material for analysis, sequencing, etc. Producing many identical copies of the same recombinant molecule is called cloning.

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Cloning can be done in vitro, by a process called the polymerase chain reaction (PCR).

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Cloning in vivo can be done in

• unicellular microbes like E. coli
• unicellular eukaryotes like yeast and
• in mammalian cells grown in tissue culture.

What are the applications of �r-DNA Technology?

1. Genetic Engineering.

2. Blotting Technique.

3. DNA sequencing.

4. DNA Chips. (Micro arrays)

5. Polymerase Chain Reaction (PCR)

6. Gene libraries - a computerized listing of known DNA sequence.

What are the applications of �r-DNA Technology?

7. Gene Bank.

8. Insulin and Diabetes control.

9. Recombinant vaccines.

10.DNA Vaccines.

11.Transgenic Plants and Animals.

12.Gene Therapy