Chapter 6 Gene Technology (Part VI)

1
Chapter review
Gene technology
Ethics
DNA recombinant technology
Gene library
Application
Genetic engineering
Gene cloning
Genomic library
cDNA library
Transgenic organism
DNA finger printing
tools
Genetically modified organism
Target gene
Cloning vector
Restriction enzyme
Modifying enzyme
Host cell
Polymerase chain reaction
methods
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At the end of the lesson, you should be able to

• Describe gene library
• List the types of gene library

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Genomic library
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Genomic library
Concept of genome

• The complete set of DNA complement of an
  organisms genes. Every species of organisms have
  its own genome that difference with another
  species

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Genomic library
genomic

• The study of whole sets of genes and their
  interactions

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Genomic library
Genomic library
A set of thousands of DNA segments from a genome,
each carried by plasmid, phage or other cloning
vector
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Genomic library

• Thousand of different recombinant plasmids are
  actually produced and a clone of each ends up in
  a colony. The complete set of thousands
  recombinant plasmid clones
• Each carrying copies of a particular segments
  from the initial genome, is refer to as a genomic
  library

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Genomic library

• Genomic library can be enormous. The 3 billion
  pairs of the human genome.
• for example, can occupy millions of plasmids,
  each harboring a small piece of DNA from one of
  our chromosomes, with many overlaps.

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Genomic library

• A genomic library also contains too much
  information for a researcher seeking a particular
  protein-encording gene, it also contains introns,
  many repeated sequences and the genes that encode
  rRNAs and tRNAs.

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Genomic library

• Gene library included a collection of cloned DNA
  fragments reprising the entire genetic material
  of an organism.
• This facilitates screening and isolation of many
  particular gene. Gene libraries are thus
  repositories of raw materials for use in genetic
  engineering.
• A human gene library may contain some 500 000
  separate clones.

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Genomic library
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cDNA library
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cDNA library
Complimentary DNA (cDNA)
A DNA molecule made in vitro using mRNA as a
template and the enzyme reverse transcriptase.
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cDNA library

• A cDNA molecule therefore corresponds to a gene,
  but lacks the introns present in the DNA of the
  genome.

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cDNA library
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cDNA library
Complimentary DNA (cDNA) library
A limited gene library using complimentary DNA.
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cDNA library

• The library includes only the genes that were
  transcribed in the cells examined Such a cDNA
  library represent only part of a cells genome.
• This is an advantage if a researcher wants to
  study the genes responsible for specialized
  functions of a particular kind or cell.

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cDNA library

• Also, by making cDNA from cells of the same type
  at different times in the life of an organism,
  one can trace changes in patterns of gene
  expression.
• cDNA represents only protein encording genes. A
  specific cDNA can be taken from cDNA library and
  used to isolated the original gene of interest
  from the genomic library.

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cDNA Insulin production
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Insulin production using cDNA

• The production of human insulin by engineered
  bacteria (E. coli ) was an important early
  example for Biotechnology.

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Insulin production using cDNA

• Once upon a time, diabetes were treated with
  regular injections of insulin from animals, like
  cattle and pig.
• But human insulin from animal sources are
  different chemically (different in only 2 of 51
  amino acids), and repeated injections from animal
  insulin led to allergic reactions in many
  patients.

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Insulin production using cDNA

• Today, human insulin produced by E. coli overcame
  this problem for many patients and actually save
  the time and cost of the treatment.
• The technique used was through recombinant DNA
  technology, using E. coli, and the name of this
  product is Humulin S.

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Insulin production using cDNA
How?
Step 1
mRNA codes insulin to be extracted from the cells
of the pancreas that produce insulin
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Insulin production using cDNA
How?
Step 2

• mRNA processes to removes introns, finally
  produce a functional mRNA

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Insulin production using cDNA
How?
Step 3
Reverse transcriptase enzyme is added, this
synthesis a cDNA by using mRNA as template. mRNA
strand then discarded/ realized by using
mRNA-degrading enzyme
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Insulin production using cDNA
How?
Step 4

• DNA polymerase enzymes is added, this synthesizes
  a second DNA strand, complementary to cDNA in
  vitro. This step produced the required gene
  (coding insulin) in cDNA double-stranded

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Insulin production using cDNA
How?
Step 5

• The DNA of gene without introns then isolated
  from it source and cut with restriction enzyme (
  Bam H1 )

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Insulin production using cDNA
How?
Step 6

• A plasmid removed from a bacterial cell is opened
  up at a specific point using the same restriction
  enzyme ( Bam H1 ).

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Insulin production using cDNA
How?
Step 7

• The DNA of gene without introns then isolated
  from it source and cut with restriction enzyme (
  Bam H1 )

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Insulin production using cDNA
How?
Step 8
The DNA is now attached to the plasmid using DNA
ligase. The resultant plasmid, carrying the
human DNA for insulin. This is called plasmid
vector/ recombinant plasmid/ rDNA.
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Insulin production using cDNA
How?
Step 9
The rDNA then reinserted into the host cell (E.
coli). Under the right conditions E. coli takes
up the rDNA by transformation.
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Insulin production using cDNA
How?
Step 10
The actual gene cloning, the production of
multiple copies of genes. The bacterium E. coli
with its recombinant plasmids, is allowed to
reproduce.
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Insulin production using cDNA
How?
Step 10
Finally, a lot of bacterial clone carrying many
copies of the gene for insulin will be produced.
Later, the bacterial can be used to produce much
insulin for health industry purposes.
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Insulin production by using cdna