Gene Cloning
A.Arputha Selvaraj
DNA Cloning: An Overview
DNA technology has launched a
revolution in Biotechnology.
DNA technology (via gene
manipulatio...
DNA Cloning Techniques
Techniques for gene cloning
enable scientists to prepare
multiple copies of DNA pieces.
DNA piece...
Recombinant DNA Technology:
A closer look
Recombinant DNA technology
requires two genes (a human
gene and a bacterial gen...
Recombinant DNA Cloning
 The process
of cloning a
human gene
in a
bacterial
plasmid can
be divided
into five
steps.
Copyr...
STEP 1. Isolation of vector and
gene-source DNA.
The source DNA comes
from human tissue cells.
The source of the plasmid...
Restriction Enzymes At Work.
Restriction
enzymes
cleave DNA at
specific sites
to isolate
genes of
interest.
Copyright © 2...
STEP 2. Insertion of DNA into
the vector.
By digesting the plasmid
and human DNA with
the same restriction
enzyme, both D...
STEP 3. Introduction of the
cloning vector into cells.
Bacterial cells take up
the recombinant
plasmids by
transformation...
STEP 4. Cloning of cells (and
foreign genes).
Plate out the
transformed bacteria
on solid nutrient
medium containing
gene...
STEP 5. Identifying cell clones
with the right gene.
In the final step, sort
through the thousands of
bacterial colonies ...
Recombinant DNA Technology:
The Big Picture
Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cumm
Thank You
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Gene cloning

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GENE CLONING

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Gene cloning

  1. 1. Gene Cloning A.Arputha Selvaraj
  2. 2. DNA Cloning: An Overview DNA technology has launched a revolution in Biotechnology. DNA technology (via gene manipulation) makes it possible to clone genes for basic research and commercial applications. DNA technology is applied to areas ranging from agriculture to criminal law.
  3. 3. DNA Cloning Techniques Techniques for gene cloning enable scientists to prepare multiple copies of DNA pieces. DNA pieces are stored in DNA libraries for easy idenfication and accessibility. DNA cloning can occur (in vitro) via Recombinant DNA Technology and Polymerase Chain Reaction (PCR).
  4. 4. Recombinant DNA Technology: A closer look Recombinant DNA technology requires two genes (a human gene and a bacterial gene) combine in vitro into one molecule (a cloning vector). The cloning vector is inserted into a bacterial cell and replicated multiple times.
  5. 5. Recombinant DNA Cloning  The process of cloning a human gene in a bacterial plasmid can be divided into five steps. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  6. 6. STEP 1. Isolation of vector and gene-source DNA. The source DNA comes from human tissue cells. The source of the plasmid is typically E. coli. The human DNA and the E. coli plasmid are cut using a restriction enzyme.
  7. 7. Restriction Enzymes At Work. Restriction enzymes cleave DNA at specific sites to isolate genes of interest. Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cummings
  8. 8. STEP 2. Insertion of DNA into the vector. By digesting the plasmid and human DNA with the same restriction enzyme, both DNA pieces can be combined easily. After mixing, both complementary pairs are joined by DNA ligase. This creates a mixture of recombinant DNA molecules.
  9. 9. STEP 3. Introduction of the cloning vector into cells. Bacterial cells take up the recombinant plasmids by transformation. This creates a diverse pool of bacteria, some bacteria that have taken up the desired recombinant plasmid DNA, other bacteria that have taken up other DNA, both recombinant and non-recombinant.
  10. 10. STEP 4. Cloning of cells (and foreign genes). Plate out the transformed bacteria on solid nutrient medium containing gene of interest and a sugar called X-gal. Only bacteria that have the gene of interest plasmid will grow. The X-gal in the medium is used to identify plasmids that carry foreign DNA.
  11. 11. STEP 5. Identifying cell clones with the right gene. In the final step, sort through the thousands of bacterial colonies to find those containing the cloned gene of interest. One technique, nucleic acid hybridization, depends on base pairing between the gene and a complementary sequence, a nucleic acid probe, on another nucleic acid molecule.
  12. 12. Recombinant DNA Technology: The Big Picture Copyright © 2002 Pearson Education, Inc., publishing as Benjamin Cumm
  13. 13. Thank You

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