Genetic Engineering/ Recombinant DNA Technology

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Genetic Engineering/ Recombinant DNA Technology

• General Genetics
• Dr. Attya Bhatti

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Genetic Engineering

• Also known as
• Gene manipulation
• Genetic modifications
• recombinant DNA technology,
• New Genetics
• Means
• altering the genes in a living organism to
  produce a Genetically Modified Organism (GMO)
  with a new genotype.
• Various kinds of genetic modification are
  possible
• inserting a foreign gene from one species into
  another,
• forming a transgenic organism
• altering an existing gene so that its product is
  changed
• Changing gene expression so that it is
  translated more often or not at all.

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History of Genetics Since 1900.
Mendelian Genetics

• 1990
• 1910
• 1920
• 1930
• 1940
• 1950
• 1960
• 1970
• 1980
• 1990
• 2000

Genetic Mapping Transformation demonstration
Microbial Genetics
Molecular Genetics

Gene Manipulation
Development of techniques Applications
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Basic Concepts of Genetic Engineering

• Recombinant DNA technology is a set of methods
  used to locate, analyze, alter, study, and
  recombine DNA sequences.
• It is used to probe the structure and function
  of genes, address questions in many areas of
  biology, create commercial products, and diagnose
  and treat diseases.

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Steps in Genetic Engineering

• Isolate the gene
• Insert it in a host using a vector
• Produce as many copies of the host as possible
• Separate and purify the product of the gene

Generation of DNA Fragments
Joining to a vector or carrier Molecule
Introduction into a host cell for amplification
Selection of required sequence.
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Step 1 Isolating the Gene
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Step 1 Alternative Method (using reverse
transcriptase)

• Reverse transcriptase
• mRNA converted into cDNA
• Complementary strand produced using DNA
  polymerase
• Advantage more mRNA in cell than DNA

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Step 2 Inserting Gene into Vector

• Vector molecule of DNA which is used to carry a
  foreign gene into a host cell

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Step 3 Inserting Vector into Host
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Replica Plating
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Step 4 Multiplication of the Host Cells by
Cloning

• Large scale fermenters by cloning
• All genetically identical because of asexual
  reproduction

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• Step 5
• Extraction of desired gene product.

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Genetically engineered corn, which produces a
toxin that kills insect pests, now comprises over
30 of all corn grown in the United States.
Recombinant DNA technology has been used to
create genetically modified crops.
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Working at the Molecular Level

• Recombinant DNA technology requires special
  methods because
• Individual genes make up a tiny fraction of the
  cellular DNA and they cannot be seen.

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Recombinant DNA Techniques

• Methods for locating specific DNA sequences
• Techniques for cutting DNA at precise locations
• Procedures for amplifying a particular DNA
  sequence billions of times, producing enough
  copies of a DNA sequence to carry out further
  manipulations
• Methods for mutating and joining DNA fragments to
  produce desired sequences
• Procedures for transferring DNA sequences into
  recipient cells

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Restriction Enzymes

• Also called restriction endonucleases that
  recognize and make double-stranded cuts in the
  sugarphosphate backbone of DNA molecules at
  specific nucleotide sequences.
• These enzymes are produced naturally by bacteria,
  where they are used in defense against viruses.
• In bacteria, restriction enzymes recognize
  particular sequences in viral DNA and then cut it
  up. A bacterium protects its own DNA from a
  restriction enzyme by modifying the recognition
  sequence, usually by adding methyl groups to its
  DNA.

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Types of Restriction Enzymes

• Three types of restriction enzymes have been
  isolated from bacteria
• Type I restriction enzymes
• Type II restriction enzymes
• Type III restriction enzymes

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Type I Restriction Enzymes

• Recognize specific sequences in the DNA
• Cut the DNA at random sites that may be some
  distance (1000 bp or more) from the recognition
  sequence

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Type II Restriction Enzymes

• Recognize specific sequences
• Cut the DNA within the recognition sequence
• Virtually all work on recombinant DNA is done
  with type II restriction enzymes

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Type III Restriction Enzymes

• Recognize specific sequences
• Cut the DNA at nearby sites
• Usually about 25 bp away

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The number of restriction sites is related to the
number of fragments produced when DNA is cut by a
restriction enzyme
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Vectors

• Is a vehicle for delivering genetic material such
  as DNA to a cell
• Cloning vectors
• Plasmid vectors
• Bacteriophage vectors

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Cloning Vectors

• is a stable, replicating DNA molecule to which a
  foreign DNA fragment can be attached for
  introduction into a cell.
• Three important characteristics
• an origin of replication which ensures that the
  vector is replicated within the cell.
• Selectable markers, which enable any cells
  containing the vector to be selected or
  identified.
• one or more unique restriction sites into which a
  DNA fragment can be inserted.

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Three characteristics of an idealized cloning
vector
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Plasmid vectors

• Plasmids are circular DNA molecules that exist
  naturally in bacteria
• contain origins of replication and are therefore
  able to replicate independently of the bacterial
  chromosome
• Used in cloning have been constructed from the
  larger, naturally occurring bacterial plasmids

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Plasmid vectors

• Example
• pUC19 plasmid
• has an origin of replication
• two selectable markersan ampicillin-resistance
  gene and a typical cloning vector

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The pUC19 plasmid is a typical cloning vector
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Bacteriophage Vectors

• Bacteriophages offer a number of advantages as
  cloning vectors.
• Most widely used bacteriophage vector is
  bacteriophage , which infects E. coli
• Advantages
• High efficiency with which it transfers DNA into
  bacteria cells

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Viewing DNA Fragments

• DNA fragments can be separated, and their sizes
  can be determined with the use of gel
  electrophoresis.
• The fragments can be viewed by
• Using a dye that is specific for nucleic acids
• By labeling the fragments with a radioactive or
  chemical tag.

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Gel electrophoresis can be used to separate DNA
molecules on the basis of their size and
electrical charge
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Cloning Genes

• Identical copies (clones) of the original piece
  of DNA are produced
• DNA fragments can be inserted into cloning
  vectors, stable pieces of DNA that will replicate
  within a cell.
• Cloning vectors must have an origin of
  replication, one or more unique restriction
  sites, and selectable markers.
• Plasmids are commonly used as cloning vectors.

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Applications

• Basic Research on gene Structure and Function
• Production of useful proteins by novel methods
• Generation of transgenic plants and animals
• Medical diagnostics and treatment

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Applications

• In addition to providing valuable new information
  about the nature and function of genes,
  recombinant DNA technology has many practical
  applications
• Include the production of pharmaceuticals and
  other chemicals, specialized bacteria,
  agriculturally important plants, and genetically
  engineered farm animals

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Applications

• Oligonucleotide Drugs
• Oligonucleotide drugs are short pieces of DNA or
  RNA that prevent the expression of particular
  genes.
• Genetic Testing
• The identification and cloning of many important
  disease causing human genes has allowed the
  development of probes for detecting
  disease-causing mutations.

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Applications

• Genetic Testing
• The identification and cloning of many important
  disease causing human genes has allowed the
  development of probes for detecting
  disease-causing mutations.
• Gene Therapy
• Ultimate application of recombinant DNA
  technology
• is gene therapy the direct transfer of genes into
  humans to treat disease

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Applications

• Gene Mapping
• Significant contribution of recombinant DNA
  technology has been to provide numerous genetic
  markers that can be used in gene mapping.
• One group of markers used in gene mapping
  comprises restriction fragment length
  polymorphisms (RFLPs, pronounced rifflips).

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Applications

• DNA Fingerprinting
• Restriction fragment length polymorphisms are
  often found in non coding regions of DNA and are
  therefore frequently quite variable in humans.