DNA Technology

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DNA Technology

• Packet 50
• Chapter 20

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Introduction

• Since the 1970s, humans have been attempted to
  manipulate and modify genes in a way that was
  somewhat predictable.

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Introduction II

• Scientists would select a gene to be inserted
  into an organism
• Cut two DNA molecules into fragments using
  restriction enzymes
• Splice the fragments together into the desired
  combination
• Producing recombinant DNA
• Introduce the new DNA into a living cell for
  replication

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Introduction III

• Recombinant DNA technology isolates and amplifies
  specific sequences of DNA by incorporating them
  into vector DNA molecules.

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The Players Involved in the Making of Recombinant
DNA
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Introduction

• Recombinant DNA is made by splicing a DNA
  fragment of interest into a small quickly
  dividing replicating molecule (plasmid).

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Donor Transgenic Organisms

• The organism providing the DNA is called the
  donor.
• After recombination, an organism that contains an
  artificially inserted, foreign piece of DNA, is
  called a transgenic organism.

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The Importance of Transgenic Organisms

• Transgenic organisms allow gene targeting and
  mutagenesis screening that help identify the
  function of a gene and its protein product.

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Vectors

• Vectors, normally in the form of plasmids, is a
  genome into which the DNA fragments, removed from
  the donor, are inserted.

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Restriction Enzymes

• Enzymes that are used to cut DNA into specific
  fragments.
• Each restriction enzyme recognizes and cuts DNA
  at a highly specific base sequence.

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The Making of Transgenic Organisms
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The Making of a Transgenic Organism

• The DNA of interest is excised, from the donor,
  using scissors known as a restriction enzyme.
• The excised DNA is called a DNA fragment.

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The Making of a Transgenic Organism

• The DNA fragment is inserted into the vector via
  one of multiple methods.

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The Making of a Transgenic Organism

• Once inserted, DNA ligase is used to join the DNA
  fragment together with the vectors genome.

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The Making of a Transgenic Organism

• The new transgenic organism is duplicated.

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Vectors Currently Under Study
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Vectors Under Study

• Vectors currently under study include
• Retroviruses
• Adenoviruses
• Herpes simplex virus
• Rhinovirus
• Human Immunodeficiency Virus (HIV)

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Genomics Genetic Libraries
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Genomic Library cDNA Library

• Genomic Library
• DNA library containing an organisms complete
  genome
• In the form of thousands of DNA fragments
• cDNA Library
• DNA library made up of DNA clones reconstructed
  using reverse transcriptase
• Must be made from mRNA
• Genomics
• Sub-discipline in genetics of characterizing the
  entire genomes of organisms.

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Homework Assignment

• What are some of the advantages, and
  disadvantages, of having a cDNA library?

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Genetic Probes
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Genetic Probes

• Genetic probes are radioactively labeled DNA or
  RNA sequence that enables geneticists to identify
  complementary nucleic acid sequences.
• If used to identify a DNA strand, the DNA
  molecule will have to be separated into into two
  strands via artificial denaturationheat.

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The Making of Genetic ProbesSouthern Blot
Technique

• DNA fragments, produced using restriction
  enzymes, are separated via gel electrophoresis.
• Fragments are blotted onto a nitrocellulose or
  nylon membrane.
• The membrane is bathed in a labeled probe for a
  specific DNA fragment.
• The selected DNA fragments are cut out of the gel

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Homework Assignment

• Define Northern Blot.
• Define Western Blot.

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Making Copies of DNA in a Lab Setting
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Introduction

• Once a sequence of DNA (DNA fragment) has been
  isolated, it is sometimes necessary to make large
  amounts of that sequence for study.

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Polymerase Chain Reaction

• Allows rapid, efficient amplification of DNA
  sequences of interest.
• In vitro technique
• Researchers target a particular DNA sequence, by
  specific primers, and then clone the DNA sequence
  by heat resistant DNA polymerase.
• Used to help amplify DNA from crime scenes and
  archaeological remains

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Gene Therapy
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Gene Therapy

• Simple ideahard to practice
• The use of sequencing, cloning and vector
  insertion techniques to deliver working versions
  of genes to individuals who are born with
  deleterious mutant versions of the gene.
• Germ Line Therapy
• Somatic Gene Therapy

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Genetic Engineering Food
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Genetic Engineering of Agricultural Species

• Foreign genes, under study, for insertion into
  commercial plant species. Helps provide
• Selective herbicide resistance
• Increased yield
• Plant-grown vaccines and pharmaceuticals
• Improved nutrient balance
• Problems?
• Human allergic reactions to foreign proteins
• Increased use of herbicides
• jumping of plasmids from commercial crops to
  weed species.
• Eco-mayhem!

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Review