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introduction
 Recombinant DNA technology or gene cloning is a new
born discipline of science which aims to alter the heredity
apparatus of a living cell.
 Recombinant DNA is a form of artificial DNA that is
made through the combination or insertion of one or
more DNA strands, therefore combining DNA
sequences as per our requirement, within different
species i.e. DNA sequences that would not normally
occur together
 It is also popularly known as genetic engineering which is
performed under highly controllable laboratory conditions
so that the cell can perform completely new functions
Basic principles of rDNA technology:
 Generation of DNA fragments & selection of
the desired piece of DNA.
 Insertion of the selected DNA into a cloning
vector to create rDNA or chimeric DNA.
 Introduction of the recombinant vectors into
host cells.
 Multiplication & selection of clones containing
the recombinant molecules.
 Expression of the gene to produce the desired
product.
3
4
5
How is Recombinant DNA made
There are three different methods by which Recombinant
DNA is made.
 Transformation
 Phage Introduction,
 Non-Bacterial Transformation.
Transformation
 The first step in transformation is to select a piece of DNA to be
inserted into a vector.
 The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA
Ligase.
 The insert contains a selectable marker which allows for
identification of recombinant molecules.
 The vector is inserted into a host cell, in a process called
transformation. One example of a possible host cell is E. Coli.
The host cells must be specially prepared to take up the foreign
DNA.
Non-bacterial Transformation
 This is a process very similar to Transformation, The only
difference between the two is non-bacterial does not use
bacteria such as E. Coli for the host.
 In micro-injection, the DNA is injected directly into the
nucleus of the cell being transformed.
 In biolistic, the host cells are bombarded with high
velocity micro projectiles, such as particles of gold or
tungsten that have been coated with DNA.
Phage Introduction
 Phage introduction is the process of transfection, which is
equivalent to transformation, except a phage is used instead of
bacteria. In vitro packaging of a vector is used. This uses lambda
or MI3 phages to produce phage plaques which contain
recombinants. The recombinants that are created can be identified
by differences in the recombinants and non-recombinants using
various selection methods.
Applications of rDNA technology
Manufacture of proteins/hormones Interferon, plasminogen
activating factor, blood clotting factors, insulin, growth hormone.
 AIDS test: Has become simple & rapid
 Diagnosis of molecular diseases: sickle cell anaemia thalassaemia,
familial hyper cholesterolaemia, cystic fibrosis
 Prenatal diagnosis: DNA from cells collected from amniotic fluid,
chorionic villi
10
Gene Therapy:
 This is achieved by cloning a gene into a vector that
will readily be taken up & incorporated into
genome of a host cell.
ADA deficiency has been successfully treated
Application in Agriculture:
 Genetically engineered plants are developed to
resist draught & diseases. Good quality of food &
increased yield of crops is also possible.
11
 Industrial Application:
Enzymes---use to produce sugars, cheese, detergents.
Protein products---used as food additives, increases nutritive value,
besides imparting flavour.
 Application in forensic medicine: The restriction analysis pattern of
DNA of one individual will be very specific(DNA fingerprinting,
but the pattern will be different from person to person. Helps to
identify criminals & to settle disputes of parenthood of children.
 Transgenesis: Gene replacement therapy will not pass on to
offspring. Therefore genes are transferred into fertilized ovum
which will be found in somatic as well as germ cells & passed on to
the successive generations.
12
principle and applications of recombinant DNA technology

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principle and applications of recombinant DNA technology

  • 1.
  • 2. introduction  Recombinant DNA technology or gene cloning is a new born discipline of science which aims to alter the heredity apparatus of a living cell.  Recombinant DNA is a form of artificial DNA that is made through the combination or insertion of one or more DNA strands, therefore combining DNA sequences as per our requirement, within different species i.e. DNA sequences that would not normally occur together  It is also popularly known as genetic engineering which is performed under highly controllable laboratory conditions so that the cell can perform completely new functions
  • 3. Basic principles of rDNA technology:  Generation of DNA fragments & selection of the desired piece of DNA.  Insertion of the selected DNA into a cloning vector to create rDNA or chimeric DNA.  Introduction of the recombinant vectors into host cells.  Multiplication & selection of clones containing the recombinant molecules.  Expression of the gene to produce the desired product. 3
  • 4. 4
  • 5. 5
  • 6. How is Recombinant DNA made There are three different methods by which Recombinant DNA is made.  Transformation  Phage Introduction,  Non-Bacterial Transformation.
  • 7. Transformation  The first step in transformation is to select a piece of DNA to be inserted into a vector.  The second step is to cut that piece of DNA with a restriction enzyme and then ligate the DNA insert into the vector with DNA Ligase.  The insert contains a selectable marker which allows for identification of recombinant molecules.  The vector is inserted into a host cell, in a process called transformation. One example of a possible host cell is E. Coli. The host cells must be specially prepared to take up the foreign DNA.
  • 8. Non-bacterial Transformation  This is a process very similar to Transformation, The only difference between the two is non-bacterial does not use bacteria such as E. Coli for the host.  In micro-injection, the DNA is injected directly into the nucleus of the cell being transformed.  In biolistic, the host cells are bombarded with high velocity micro projectiles, such as particles of gold or tungsten that have been coated with DNA.
  • 9. Phage Introduction  Phage introduction is the process of transfection, which is equivalent to transformation, except a phage is used instead of bacteria. In vitro packaging of a vector is used. This uses lambda or MI3 phages to produce phage plaques which contain recombinants. The recombinants that are created can be identified by differences in the recombinants and non-recombinants using various selection methods.
  • 10. Applications of rDNA technology Manufacture of proteins/hormones Interferon, plasminogen activating factor, blood clotting factors, insulin, growth hormone.  AIDS test: Has become simple & rapid  Diagnosis of molecular diseases: sickle cell anaemia thalassaemia, familial hyper cholesterolaemia, cystic fibrosis  Prenatal diagnosis: DNA from cells collected from amniotic fluid, chorionic villi 10
  • 11. Gene Therapy:  This is achieved by cloning a gene into a vector that will readily be taken up & incorporated into genome of a host cell. ADA deficiency has been successfully treated Application in Agriculture:  Genetically engineered plants are developed to resist draught & diseases. Good quality of food & increased yield of crops is also possible. 11
  • 12.  Industrial Application: Enzymes---use to produce sugars, cheese, detergents. Protein products---used as food additives, increases nutritive value, besides imparting flavour.  Application in forensic medicine: The restriction analysis pattern of DNA of one individual will be very specific(DNA fingerprinting, but the pattern will be different from person to person. Helps to identify criminals & to settle disputes of parenthood of children.  Transgenesis: Gene replacement therapy will not pass on to offspring. Therefore genes are transferred into fertilized ovum which will be found in somatic as well as germ cells & passed on to the successive generations. 12