Recombinant DNA Recombinant DNA TechnologyTechnology

Tapeshwar Yadav(Lecturer)BMLT, DNHE, M.Sc. Medical Biochemistry

A – A good student is liked by teacher

G – Greets everyone with smileO – ObedientO – On time for collegeD – Dresses neatly

S – Studies with interestT – Treats everyone with smileU – Understands everythingD – Does daily home workE – Eager to know new thingsN – Never misbehavesT – Talks little in class

Biotechnology may be defined as “the method by which a living organism or its parts are used to change or to incorporate a particular character to another living organism”

It involves the application of scientific principles to the processing of materials by biological agents.

Genetic recombination is the exchange of information between two DNA segments.

This is a common occurrence within the same species.

But by artificial means, when a gene of one species in transferred to another living organism, it is called recombinant DNA technology.

In common, this is known as genetic engineering.

Definition of recombinant Definition of recombinant DNADNA

• Production of a unique DNA molecule by joining together two or more DNA fragments not normally associated with each other

• DNA fragments are usually derived from different biological sources

Definition of recombinant Definition of recombinant DNA technologyDNA technology

• A series of procedures used to recombine DNA segments. Under certain conditions, a recombinant DNA molecule can enter a cell and replicate.

History of recombinant History of recombinant DNA technologyDNA technology

• Recombinant DNA technology is one of the recent advances in biotechnology, which was developed by two scientists named Boyer and Cohen in 1973.

Development of Development of molecular biologymolecular biology

• Early research on prokaryotic genetics and the development of molecular techniques has led to a new discipline called MOLECULAR BIOLOGY

• “Tools” have been developed (and still continue to be modified/improved) to enable scientists to examine very specific regions of the genome or genes.

• Advances in Molecular Biology– The combination of

restriction/modification enzymes and hybridization techniques enable the application of a wide variety of procedures

TECHNIQUES and PROCEDURES

• Gene isolation/purification/synthesis• Sequencing/Genomics/Proteomics• Polymerase chain reaction (PCR)• Mutagenesis (reverse genetics)• Expression analyses (transcriptional and translational

levels)• Restriction fragment length polymorphisms (RFLPs)• Biochemistry/ Molecular modeling• Gene therapy

Applications

• Quantitative preparation of biomolecules• Recombinant Vaccines• Antenatal diagnosis of genetic diseases• Monoclonal antibodies• Cell/tissue culture• To identify mutations in genes• Xenotransplantation

Contd…Contd…

• To detect activation of oncogenes • Production of next generation antibiotics• Forensics• Biosensors• Genetically modified crops• Bioterrorism detection

1.Quantitative Preparation of 1.Quantitative Preparation of BiomoleculesBiomolecules

• If molecules are isolated from higher organisms, the availability will be greatly limited.

• For eg.- To get 1 unit of growth hormone, more than 1000 pituitaries from cadavers are required.

• By means of recombinant technology, large scale availability is now assured.

2. Risk of contamination is Eliminated2. Risk of contamination is Eliminated

• It is now possible to produce a biological substance without any contamination.

• Hepatitis, caused by HBV, is highly contagious.

• Preparations of vaccines or clotting factors are free from contaminants such as hepatitis B particles.

• RD-Technology provides the answer to produce safe antigens for vaccine production.

3. Specific probes for Diagnosis of 3. Specific probes for Diagnosis of DiseasesDiseases

Specific probes are useful for:

i. Antenatal diagnosis of genetic diseases.

For eg.- many of the single gene defects like cystic fibrosis, phenyl ketonuria etc.

Could be identified by taking cell samples from fetus.

ii. To identify viral particles or bacterial DNA in suspected blood and tissue samples.

Contd…Contd…

iii. To demonstrate virus integration in transformed cells.

iv. To detect activation of oncogenes in cancer.

v. To pinpoint the location of a gene in a chromosome.

vi. To identify mutations in genes.

4. Gene Therapy4. Gene Therapy

• It is an important applications of RD-Technology• Normal genes could be introduced into the patient so

that genetic diseases can be cured.

• How to find one gene in large genome? • A gene might be 1/1,000,000 of the genome.

Three basic approaches: • 1. Cell-based molecular cloning: create and

isolate a bacterial strain that replicates a copy of your gene.

• 2. Polymerase chain reaction (PCR). Make many copies of a specific region of the DNA.

• 3. Hybridization: make DNA single stranded, allow double strands to re-form using a labeled (e.g. radioactive) version of your gene to make it easy to detect.

Basic principle of recombinant DNA Basic principle of recombinant DNA technologytechnology

• The DNA is inserted into another DNA molecule called ‘vector’

• The recombinant vector is then introduced into a host cell where it replicates itself, the gene is then produced

Cell-Based Molecular Cell-Based Molecular CloningCloning

• The original recombinant DNA technique: 1974 by Cohen and Boyer.

• • Several key players:• 1. restriction enzymes. Cut DNA at specific

sequences. e.g. EcoR1 cuts at GAATTC and BamH1 cuts at GGATCC.

• – Used by bacteria to destroy invading DNA: their own

DNA has been modified (methylated) at the corresponding sequences by a methylase.

•

2. Plasmids: independently replicating DNA circles (only circles replicate in bacteria). Foreign DNA can be inserted into a plasmid and replicated.

– Plasmids for cloning carry drug resistance genes

that are used for selection.– Spread antibiotic resistance genes between bacterial

species 3. DNA ligase. Attaches 2 pieces of DNA together. 4. transformation: DNA manipulated in vitro can be put

back into the living cells by a simple process .– The transformed DNA replicates and expresses its

genes.

Best Way to keep COOL in Best Way to keep COOL in SUMMERSUMMER ? ! ? ? ! ?

II. Restriction EndonucleasesII. Restriction Endonucleases

A. Origin and functionA. Origin and function

• Bacterial origin = enzymes that cleave foreign DNA

• Named after the organism from which they were derived– EcoRI from Escherichia coli– BamHI from Bacillus amyloliquefaciens

• Protect bacteria from bacteriophage infection– Restricts viral replication

• Bacterium protects it’s own DNA by methylating those specific sequence.

B. AvailabilityB. Availability

• Over 200 enzymes identified, many available commercially from biotechnology companies

C. ClassesC. Classes

• Type I– Cuts the DNA on both strands but at a non-

specific location at varying distances from the particular sequence that is recognized by the restriction enzyme

– Therefore random/imprecise cuts– Not very useful for rDNA applications

• Type II– Cuts both strands of DNA within the

particular sequence recognized by the restriction enzyme

– Used widely for molecular biology procedures

– DNA sequence = symmetrical

• Reads the same in the 5’ 3’ direction on both strands = Palindromic Sequence

• Some enzymes generate “blunt ends” (cut in middle)

• Others generate “sticky ends” (staggered cuts)

– H-bonding possible with complementary tails– DNA ligase covalently links the two fragments

together by forming phosphodiester bonds of the phosphate-sugar backbones

DNA Ligase in Action!DNA Ligase in Action!

III. Vectors for Gene III. Vectors for Gene CloningCloning

“To be good & to do good that is the whole of religion”