Presentation Recombinant dna technology Presented by: shalini negi

Intentionally modifying genomes of organisms, by natural and artificial processes, for practical Purposes.

Recombinant DNA molecule:

A vector (plasmid /phage/ virus) into which the desired DNA fragment has been inserted by use of specific enzymes to enable its cloning in an appropriate host(bacteria).

Three Goals of Recombinant DNA Technology:

1. Eliminate undesirable phenotypic traits in humans, animals, plants and microbes. 2. Combine beneficial traits of two or more organisms to create valuable new organisms.

3. Create organisms that synthesize products of humans need.

Objective:
Isolation of multiple copies of a desired gene and transfer it to suitable organism to achieve:

Large scale production of protein encoded by the gene. Expression of the gene leading to development of desired phenotype. Know the basics of gene structure, function and regulation. Be familiar with the basic methods of molecular genetics. Understand the meaning of DNA sequence and amino acid polymorphisms. The main purpose of rDNA technology is to make large amounts of DNA (gene) &/or its Proteins. (Ex: Insulin).

Recombinant DNA Process Example:

DNA Cloning COMPONENTS:

Vector Plasmid or virus. Should have: Antibiotic Resistance gene & Lac Z gene for color selection.

DNA or Gene to Clone

Restriction Enzyme DNA Ligase

Antibiotics
Colorless X-galactose dye A host, usually bacteria.
Plasmid Vector with AmpR-gene.

DNA Cloning INVOLVES 5 STEPS:

1. 2. 3. 4. 5. Restriction Enzyme Digestion. Ligation. Transformation of Host. Growth of Transformant. Selection for recombinant.

Steps in Recombinant DNA Technology 1. Restriction Enzyme Digestion:

Digest the DNA to Clone & the Vector with Same Restriction Enzyme. Ex: EcoRI, To generate complementary Sticky ends that can base pair.

Steps in Recombinant DNA Technology 2. Ligation:

DNA Ligase seals the ends of DNA to clone & the vector DNA.
The DNA now inserted into Vector DNA = Recombinant DNA
DNA insert
LacZ gene

Vector
AmpR
Recombi nant Plasmid DNA

Ligation of sticky ends of vector and DNA insert

Steps in Recombinant DNA Technology 3. Transformation of Host:

Recombinant DNA plus all other ligation products are mixed with host solution. Chemicals are used to activate uptake by host. Host with vector or recombinant DNA is called Transformant (transformed host). Host will now become antibiotic resistant.

Steps in Recombinant DNA Technology

Transformation Results:
Some hosts will not uptake anything.

Some will uptake vector alone. Some will take Recombinant DNA.

Steps in Recombinant DNA Technology 4. Growth of Transformant: Agar Plate with

Ampicillin + X-Gal

Transformants and other products are cultured in a dish containing: Nutrients. Recombinant l virions form plaques a colorless dye called X-galactose. LacZ breaks down this dye forming blue color. Transformants containing plasmid or recombinant plasmid DNA will form COLONIES (bumps) on the dish. Transformants containing virus vector or recombinant viral DNA will from PLAQUES (dents). Antibiotic.

Steps in Recombinant DNA Technology 5.Selection for Recombinant:

X-galactose in nutrient dish allows selection of successful ligation of DNA insert that: Inactivation of LacZ gene. White color (no blue color).
Ampicillin in nutrient allows selection of successful transformation.

Agar dish after recombinant DNA Technology. Shows both blue and white (clear) colonies.

Analysis of Recombinant DNA Technology ie.Results & Selection for Recombinant:

In the example shown above, the b-galactosidase

gene is inactivated. The substrate "X-gal" turns blue if the gene is intact, ie. makes active enzyme. White colonies in X-gal imply the presence of recombinant DNA in the plasmid.

Recombinant DNA Analysis

Polymerase Chain Reaction (PCR) With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. Each PCR cycle involves three steps: 1. Denaturation (high temperature) 2. Annealing of primers (low temperature) 3. DNA synthesis (intermediate temperature) -Taq polymerase (DNA polymerase)

Recombinant DNA Applications: 1. Recombinant DNA Vaccines:

Strategy for a subunit vaccine for herpes simplex

Agricultural Applications

Ti (tumor-inducing) plasmid is the most used vector for plant genetic engineering -Obtained from Agrobacterium tumefaciens, which normally infects broadleaf plants -However, bacterium does not infect corn, rice and wheat
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SUMMARY:
Recombinant technology begins with the isolation of a gene of interest. The gene is then inserted into a vector and cloned. The gene of interest (foreign DNA) is integrated into the plasmid or phage, and this is referred to as recombinant DNA. Once the vector is isolated in large quantities, it can be introduced into the desired host cells such as mammalian, yeast, or special bacterial cells. The host cells will then synthesize the foreign protein from the recombinant DNA. When the cells are grown in vast quantities, the foreign or recombinant protein can be isolated and purified in large amounts.

Applications of recombinant DNA technology

Site-specific mutagenesis Analysis of DNA polymorphisms Testing for human genetic disease mutations Isolation of human genes DNA typing Analysis of expression of individual genes Analysis of protein-protein interactions Gene therapy Biotechnology and commercial products Genetic engineering of plants Structural genomics Functional genomics Comparative genomics Ethics and the Human Genome Project