Molecular Cloning or Genetic Engineering or Recombinant DNA Technology:

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Molecular Cloning or Genetic Engineering or
Recombinant DNA Technology To clone means to
make identical copies. DNA cloning involves
separating a specific gene or DNA segment from a
larger chromosome, attaching it to a small
carrier DNA. The resultant hybrid DNA is called
recombinant DNA, which is transferred to a proper
host (bacteria, virus or yeast) and replicated to
make multiple copy of the selected gene. When
cloned under an appropriate expression vector, a
gene can be expressed (I.e. transcribed and
translated), at desired level to produce
recombinant proteins. This technology has made
it possible to isolate, clone and produce DNA for
all the genes in appropriate quantity so that
they can be sequenced and characterized.
Similarly, some of the genes which are expressed
at very low level, can be cloned and desired
amount of recombinant proteins can be produced.

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• Five steps of cloning
• Cutting the DNA to be cloned from the chromosomal
  using sequence specific Restriction Endonuclease.
• Selecting a cloning vector (a small molecule
  capable of self-replicating inside host cells),
  and cutting the cloning vector with the same
  restriction endonuclease producing the cohessive
  ends.
• Incubating the vector and subject DNA togather to
  aneal and then joining them using DNA ligase. The
  resultant DNA is called recombinant DNA.
• Transferring the reconbinant DNA to an
  appropriate host such as bacteria, virus or yeast
  which will provide necessory biomachinary for DNA
  replication.
• Identifying the host cells that contain the
  recombinant DNA.

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Cloning Vectors Circular plasmid DNAs are the
most common cloning vectors. These are 1 to 200
kb long DNA duplexes containing required genetic
machinery such as replication origin to permit
their autonomous propagation in host cell. The
plasmid vectors contain some specific genes
responsible for antibiotic resistance, which can
be used to select the bacterial colonies
containing recombinant plasmids. In order to
clone the foreign DNA at specific site, a
synthetic oligonucleotide containing restriction
sites for several REs is ligated in the plasmid.
This region is reffered as polylinker
region. Bacteriophage-based cloning
vectors Yeast artificial chromosomes vectors
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Joining of two DNA fragments Dale Kaiser and
Paul Berg Used terminal deoxynucleotidyl
transferase (TdT or terminal transferase) to
generate sticky or cohesive ends in the DNA. TdT
is a mammalian enzyme which adds nucleotide to
the 3-OH group of DNA without any requirement of
primer. The two DNA fragments to be joined, are
subjected to TdT reaction in the presence of dTTP
and dATP separately to add poly-T to one DNA and
poly-A to other DNA. The two DNA fragments with
cohesive ends are annealed, the gaps are filled
with DNA polymerase and then they are joined
covalently by ligase. TdT requires at least
three nucleotides free at 3 end (I.e unpaired),
it can be created by bacteriophage lambda
exonuclease.
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A constructed E. Coli plasmid pBR322 designed
specially for cloning in E. Coli. A foreign gene
cloned in PstI restriction site can be selected
as depicted in next slide.
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Selection of the bacterial colony containing
recombinant DNA by antibiotic resistance and
sensitivity.
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Bacteriophage l cloning vector This virus is
very efficient in delivering its 48kb long DNA
into a host bacterium. One third of its DNA is
non-essential and can be replaced by foreign
DNA. The recombinant DNA can be packaged into
phage particle by adding this DNA to bacterial
extract containing proteins for packaging.
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• Construction of complementary DNA (cDNA) library
• cDNAs are the DNA with complementary sequence to
  mRNA. The cDNA represents genes expressed at mRNA
  level.
• mRNA is isolated using oligi-dT column and
  annealed with oligo-dT primer.
• cDNA is generated by reverse transcriptase and
  dNTPs
• The mRNA is degraded by alkaline hydrolysis and a
  double standed DNA is prepared using DNA
  polymerase-I and dNTPs.
• The cDNAs created in this way are cloned in
  appropriate plasmid or phage vector and
  tranfected to host cells.

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• Amplification of a DNA segment by Polymerize
  Chain Reaction (PCR)
• DNA strands are separated by heating.
• Cool the DNA and add synthetic oligonucleotide
  primers that flank the region to be amplified.
• Add thermostable DNA polymerase (Taq1 polymerase)
  to catalyse 5-3 synthesis of DNA.
• Repeat steps 1, 2 and 3 30 to 40 times to
  generate thousands to millions of copies of the
  original DNA.

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• Colony hybridization to screen the bacterial
  plasmid library
• The cDNA library (bacteria containing different
  cDNAs) is plated on agar plates in appropriate
  media.
• A nitrocellulose paper is pressed upon the the
  bacterial colonies. Some bacteria are transferred
  to NC paper.
• The NC is treated with alkali to lyse the cells
  and expose the cDNAs.
• The DNA binds to NC paper, and a radioactive DNA
  probe corresponding to the desired gene is used
  to hybridize with the NC paper.
• DNA from the Colonies with the desired gene will
  be seen on X-ray film after the exposure of the
  hybridized NC paper.

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• DNA Microarray analysis for gene expression
• DNA chips conatining spots with the DNA of known
  genes are available comme-rcially, or one can
  make one with desired DNAs.
• mRNA is isolated from control and diseased tissue
  and cDNA is made using different fluorescent
  nucleotide for the two mRNAs.
• The fluorescent cDNAs is then used for
  hybridization with the DNA microarray chip.
• The fluorescent spots indicate the expression of
  corresponding gene.

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• Site-directed Mutagenesis
• Michael Smith (Canada) was awarded with noble
  prize for his work on site directed mutagenesis.
• One can synthesize a mutated DNA and insert into
  the gene using restriction enzyme and ligase.
• The most poular proceedure involves working with
  single stranded DNA. An oligonucleotide is made
  with desired single nucleotide change and then
  used as a primer with DNA polymerase to make a
  mutant copy of genes.

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Creation of recombinant plant using a plant
parasite agrobacterium and two plasmid stretegy.
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A tobacco plant in which the gene for fire fly
luciferase is expressed the plant glows when
watered with luciferin (a substrate for this
enzyme).
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Tomato plants engineered to be resistant to some
insect larvae
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