RECOMBINANT DNA

TECNOLOGY
It is a technique used in genetic engineering. It involves the
identification, isolation and insertion of gene of interest(DNA) into a
vector DNA to form a recombinant DNA molecule so that, a large
quality of gene of interest or product encoded by that gene can be
produced when that recombinant DNA is introduced into a host cell

Recombinant DNA is also know as chimeric DNA. This name is

inspired from the fact that chimera is a monster in Greek mythology
that has lions head, a goats body and a serpents tail. recombinant
DNA is obtained by inserting the DNA of interest into a vector DNA
DNA
• PRINCIPLE
Every protein is encoded by a certain gene. If the gene can be
identified, then that can be used to produce a large quantity of the
same gene or product encoded by that gene. The gene of interest can
be transfered to a vector DNA to produce recombinant DNA or cloned
gene. Vectors are self-replicating inside an appropriate host cell.
Introduction of recombinant vector into host cells cause multiplication
of the cloned gene. Expression of the cloned gene
produced desired protein.
 PROCESS…
PROCESS..
The recombinant technology involves
1. Generation of DNA fragments and selection of the desired piece
of DNA
2. Insertion of the selected DNA into a cloning vector to produce
recombinant DNA
3. Introduction of the recombinant DNA into host cells(eg. bacteria)
4. Multiplication of clones containing the recombinant DNA
molecules
5. Selection of clones containing the recombinant DNA
6. Expression of the gene to produce the desired product
7. Separation and purification of the product
• The desired piece of DNA can also be obtained from
1. Genomic library
2. cDNA library
3. Chemical synthesis

1.The genomic library is a collection of the total genomic DNA from a single organism.
The DNA is stored in a population of identical vectors, each containing a different
insert of DNA. A cDNA library represents a collection of only the genes that are
encoded into proteins by an organism. Complementary DNA ,or cDNA, is created
through reverse transcription of messenger RNA and a library of cDNAs is generated
using DNA cloning technology. In chemical synthesis, a DNA strand is synthesized from
the nucleotides without using template strand and DNA polymerase.
DNA MODIFYING ENZYMES INVOLVED
IN RECOMBINANT DNA
TECHNOLOGY
1. Restriction endonuclease enzyme (DNA cutting enzyme)
Nucleases are the enzymes that break the phospodiaster bonds (that hold nucleotides
together)of DNA.endonucleases act on the internal phospodiaster bonds while
exonucleases degrade DNA from the terminal ends.some of the endonucleases cut DNA
nonspecifically at any site.but some endonucleases cut DNA at specific nucleotide
sequences. the letter endonucleases are the know as restriction endonucleases or
restriction enzymes

2.DNA ligases
The cut DNA fragments are covalently joined together by DNA ligases. These enzymes
were originally isolated from viruses.they also occur in E coli and eukaryotic cells.
VECTORS the cloning vehicles:
• Vectors are the DNA molecules,which can carry a foreign DNA fragment to be
cloned. they are self-replicating in an appropriate host cell.the most
important vectors are
• Plasmids
Bacteriophages
• Cosmids
• Phasids
Ideal vectors:
• It should be small in size.
• It should have a single restriction endonuclease site.
• It should have an origin of replication.
• It should have 1-2 genetic markers.
 Application of recombinant DNA technology

1)Medicines:
• Production of antibiotics like pencillins,streptomycin,etc
• Production of hormones like insulin, human growth hormone, human
chorionic gonodotrophin, erythropoietin, ect
• Production of vaccines like hepatitis B,herpes, influenza,etc
• Production of interferon and iterleukins
• Production of enzymes like urokinase,tissue plasminogen activator
• Production of factor 7,a blood clotting factor for haemophilia A
• Production of monoclonal antibodies against cancer,AIDS, etc
• Production of transgenic animals: animals which carry foreign genes
are called transgenic animals. Example cow,sheep,goat.
2)INDUSTRY
1. Production of chemical compounds of commercial importance.
2. Improvement of existing fermentation process.
3. Production of proteins from wastes.

3)PLANTS
4. Distant hybridization transfer of genes between distantly related species.
5. Development of transgenic plants.
6. Development of root nodules in cereal crops.
7. Development of c4 plants with high photosynthetic rate and higher
potential of biomass production than c3 plants.
GENE THERAPHY
Gene therapy is a precess of inserting gene into cells to treat diseases.it
primarilly involves genetic manipulation in animals or humans to
correct a disease and keep the organism in good health
Approaches for gene therapy
There are two approaches to achieve gene theraphy
1 somatic cell gene therapy.
2 germ cell gene therapy.
1=somatic cell therapy
• The non reproductive (non-sex) cells of an organism are referred to as somatic
cells.these are the cells of an organism other than sperm or egg cells e.g bone marrow
cells,blood cells ,skin cells,intestinal cells,etc. somatic cell gene therapy involves the
insertion of a fully function and expressible gene into a target somatic cell to correct a
genetic disease permanently

•2=Germ cell gene therapy

• The reproductive ( sex) cells of an organism constitute germ cell line.gene therapy
involving the introduction og DNA into germ cells is passed on to the successive
generations. The genetic alterations in somatic cells,are not carried to the next
generations. So at present, all the research on gene therapy is directed to correct the
genetic defects in somatic present,all the research on gene therapy is directed to
correct the genetic defects in somatic cells.
Development of gene therapy
• Development of gene therapy in human for any specific disease involves the
following steps:
1. In- vitro experiments and research on laboratory animals (pre-clinical trials).
2. Phase 1 trials with a small number(5-10)of human subjects to safety of the
product
3. Phase 2 trials with more human subjects to assess whether the product is
helpful
4. Phase 3 trials in large human sample for a final and comprehensive analysis
of the safety and efficacy of the product
Types of gene therapy
1. Ex-vivo gene therapy
2. In-vivo gene therapy

Ex-vivo gene therapy:

It can be applied to only selected tissues(e.g.bone marrow,hepatocytes)whose cells can be
cultured in the laboratory.it involves following steps:
3. Isolate cells with genetic defects from a patient.
4. Grow the cells in culture.
5. Introduce the therapeutic gene to correct gene defect
6. Select the genetically corrected cells(stable transformants)and grow.
7. Transplant the modified cells to the patient.
It basically involves the use of patients cells of culture and genetic correction and then their
return back to the patient.
In-vivo gene therapy

The direct delivery of the therapeutic gene(DNA) into the target cells of
a particular tissue of a patient constitutes in-vivo gene therapy.many
tissue are the potential candidates for this approaches.these include
liver,muscle,skin,spleen,lung,brain and blood cells
The succeses of in-vivo gene therapy mostly depends on the following
parameters:
• The efficiency of the uptake of the remedial(therapeutic)gene by the
target cells.
• Intracellular degradation of the gene and its uptake by nucleus.
• The expression capability of the gene.
 Cell culture

• Cell culture is the process by which cells are grown under controlled
conditions,generally outside their environment(in-vitro). Tissue and organ
can also be cultured in-vitro. cell culture is simple.organ culture is
complicated and difficult to establish.

• Culture environment
following media are needed for cell survival:
1. Substrate(solid and liquid suspension)
2. Nutrients (energy source)
3. Environmental factors
4. Sterility
5. The physiological and physiochemical conditions are provided/controlled
Culture media
• Media provides necessary nutrients,hormones and growth factors for
cell growth as well as regulating the PH and osmotic pressure of the
culture.
• Serum is a source for hormones,minerals,growth
factors,lipids,enzymes,micronutrients,trace elements,etc.media are
three types based on the requirements for supplementation with
serum

• Basal media
• Reduced serum media
• Serum free medium
Types of cell culture
• The cell culture can be either primary or secondary.
1. Primary cell culture
• Adherent culture
• Suspension culture

2.Secondary culture
• finite cell line
Continuous cell line
Primary cell culture
• These are the first cells taken from an organism.the cells are
dissociated from parent tissue(e.g.liver or kidney) using mechanical
method/enzymatic method.then they are grown in-ivtro in culture
medium(artificial)using suitable glass/plastic containers.
• TYPES;
• A)Adherent cell culture or anchorage dependent cells: these cells
require an attachment for growth.the cells are derived from immobile
organs like kidney,liver etc. the cells grow on the surface of a solid
meida also known as monolayer.it is easy to harvest on any
surfaces.so,growth is easy.but we get a small number of cells as
growth is halted after a single layer.
Secondary cell culture
• Primary cell culture subculture secondary culture
• Subculture refers to transfer of cells from one culture to another culture vessel.
• This is periodically required to provide fresh nutrients , provide space for
continuously growing cell lines. When a primary culture is subcultured, it
becomes secondary subculture.
• After at least one sub culture, cell lines are produced. The secondary cell
culture (cell lines) can be finite or continuous. Finite cell culture grows and
eventually die. continuous cell culture is also known as transformed cell culture.
It is done by in-vitro transformation of finite cells. Hela cells are examples of
continuous cell lines. Hela is the oldest and most commonly used human cell
line.