MUHAMMAD UMAR : Lecturer In
Zoology GDC Shabqadar Charsadda
Recombinant DNA Technology
Introduction
 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
Insulin Isolation from Pigs
 Before 1978, insulin was extracted from the pancreas
glands of slaughtered pigs.
 Process: Pig pancreases were collected, ground up, and
treated with acidified alcohol to extract insulin.
 Purification: The mixture was subjected to multiple
chemical processes and crystallization, including high-
performance liquid chromatography (HPLC), to remove
impurities, though some contaminating substances often
remained.
 Yield: It took approximately two tons of animal pancreas
tissue to produce 8 ounces (about 227 grams) of purified
insulin
Drawbacks of Pig Derived Insulin
 Position: The substitution occurs at the carboxyl-terminal
(C-terminal) of the B-chain, which is position B30.
 Amino Acid Change: Pig insulin has Alanine (Ala),
whereas human insulin has Threonine (Thr).
 Effect: The substitution of a hydrophobic amino acid
(Alanine) in pig insulin for a hydrophilic one (Threonine) in
humans can make pig insulin slightly less soluble and
slightly slower acting, although it has historically been used
to treat diabetes.
 Immunogenicity: Because of this single amino acid
difference, pig insulin can, in rare cases, trigger an immune
response, although it is very close to human insulin.
Historical Background
 Recombinant DNA (rDNA) technology, developed
in the early 1970s, originated from the discovery
of restriction enzymes and the ability to splice
DNA. Key milestones include Paul Berg
producing the first recombinant molecule in
1972, and Stanley Cohen and Herbert Boyer
creating the first transgenic organism (using E.
coli) in 1973.
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.
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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
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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.
14
 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.
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Recombinant DNA Technology.pptx Advace slides for BS level

Recombinant DNA Technology.pptx Advace slides for BS level

  • 1.
    MUHAMMAD UMAR :Lecturer In Zoology GDC Shabqadar Charsadda Recombinant DNA Technology
  • 2.
    Introduction  Recombinant DNAis 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.
    Insulin Isolation fromPigs  Before 1978, insulin was extracted from the pancreas glands of slaughtered pigs.  Process: Pig pancreases were collected, ground up, and treated with acidified alcohol to extract insulin.  Purification: The mixture was subjected to multiple chemical processes and crystallization, including high- performance liquid chromatography (HPLC), to remove impurities, though some contaminating substances often remained.  Yield: It took approximately two tons of animal pancreas tissue to produce 8 ounces (about 227 grams) of purified insulin
  • 4.
    Drawbacks of PigDerived Insulin  Position: The substitution occurs at the carboxyl-terminal (C-terminal) of the B-chain, which is position B30.  Amino Acid Change: Pig insulin has Alanine (Ala), whereas human insulin has Threonine (Thr).  Effect: The substitution of a hydrophobic amino acid (Alanine) in pig insulin for a hydrophilic one (Threonine) in humans can make pig insulin slightly less soluble and slightly slower acting, although it has historically been used to treat diabetes.  Immunogenicity: Because of this single amino acid difference, pig insulin can, in rare cases, trigger an immune response, although it is very close to human insulin.
  • 5.
    Historical Background  RecombinantDNA (rDNA) technology, developed in the early 1970s, originated from the discovery of restriction enzymes and the ability to splice DNA. Key milestones include Paul Berg producing the first recombinant molecule in 1972, and Stanley Cohen and Herbert Boyer creating the first transgenic organism (using E. coli) in 1973.
  • 6.
    Basic principles ofrDNA 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. 6
  • 7.
  • 8.
  • 9.
    How is RecombinantDNA made There are three different methods by which Recombinant DNA is made.  Transformation  Phage Introduction,  Non-Bacterial Transformation.
  • 10.
    Transformation  The firststep 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.
  • 11.
    Non-bacterial Transformation  Thisis 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.
  • 12.
    Phage Introduction  Phageintroduction 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.
  • 13.
    Applications of rDNAtechnology 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 13
  • 14.
    Gene Therapy:  Thisis 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. 14
  • 15.
     Industrial Application: Enzymes---useto 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. 15