Introduction to

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Introduction to Studying DNA
Chapter 4
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Learning Outcomes

• Describe the structure and function of DNA and
  explain the process by which it encodes for
  proteins
• Differentiate between eukaryotic and prokaryotic
  chromosomal structure and explain how this
  difference impacts gene regulation in the two
  cell types
• Differentiate between bacterial cultures grown in
  liquid and solid media and explain how to prepare
  each media type using sterile technique
• Discuss the characteristics of viruses and their
  importance in genetic engineering
• Explain the fundamental process of genetic
  engineering and give examples of the following
  applications recombinant DNA technology,
  site-specific mutagenesis, and gene therapy
• Describe the process of gel electrophoresis and
  explain how the characteristics of molecules
  affect their migration through a gel

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4.1 DNA Structure and Function
The manipulation of genetic information, DNA and
RNA codes, is at the center of most biotechnology
research and development.
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The Central Dogma of Biology. Proteins are
produced when genes on a DNA molecule are
transcribed into mRNA, and mRNA is translated
into the protein code. This is called gene
expression. At any given moment, only a
relatively small amount of DNA in a cell is being
expressed.
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DNA Structure. The nucleotides in one chain of
the helix face one direction, while those in the
other strand face the other direction. Each
nucleotide contains a sugar molecule, a phosphate
group, and a nitrogenous base. Nitrogenous bases
from each strand bond to each other in the center
through H-bonds. The H-bonds are rather weak
therefore, the two strands of DNA separate easily
in high temperatures.
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Similarities in DNA Molecules Among Organisms

• All DNA molecules are composed of four nucleic
  monomers
• Adenosine deoxynucleotide (A)
• Cytosine deoxynucleotide (C)
• Guanosine deoxynucleotide (G)
• Thymine deoxynucleotide (T)
• Virtually all DNA molecules form a double helix
• The amount of adenosine equals the amount of
  thymine
• The amount of guanosine equals the amount of
  cytosine
• Nucleotides in each strand are oriented in the
  opposite direction of the other strand
• Nitrogenous bases
• DNA undergoes semiconservative replication

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DNA Replication. DNA replicates in a
semiconservative fashion in which one strand
unzips and each side is copied. It is considered
semiconservative since one copy of each parent
strand is conserved in the next generation of DNA
molecules.
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Variations in DNA Molecules

• The number of DNA strands in the cells of an
  organism
• The length in the base pairs of the DNA strands
• The number and type of genes and noncoding
  regions
• The shape of the DNA strands

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Vocabulary

• Chromatin nuclear DNA and proteins
• Gene a section of DNA on a chromosome that
  contains the genetic code of a protein
• Nitrogenous base an important component of
  nucleic acids (DNA and RNA), composed of one of
  two nitrogen-containing rings forms the critical
  hydrogen bonds between opposing strands of a
  double helix
• Base pair two nitrogenous bases that are
  connected by a hydrogen bond for example, an
  adenosine bonded to a thymine or a gaunine bonded
  to a cytosine
• Phosphodiester bond a bond that is responsible
  for polymerization of nucleic acids by linking
  sugars and phosphates of adjacent nucleotides
• Hydrogen bond a type of weak bond that involved
  the sandwiching of a hydrogen atom between two
  fluorine, nitrogen, or oxygen atoms especially
  important in the structure of nucleic acids and
  proteins
• Pyrimidine a nitrogenous base composed of a
  single carbon ring a component of DNA
  nucleotides
• Purine a nitrogenous base composed of a double
  carbon ring a component of DNA nucleotides
• Antiparallel a reference to the observation
  that strands on DNA double helix have their
  nucleotides oriented in the opposite direction to
  one another
• Semiconservative replication a form of
  replication in which each original strand of DNA
  acts as a template, or model, for building a new
  side in this model one of each new copy goes
  into a newly forming daughter cell during cell
  division

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4.1 Review Questions

1. Describe the relationship between genes, mRNA,
   and proteins.
2. Name the four nitrogen-containing bases found in
   DNA molecules and identify how they create a base
   pair.
3. The strands on a DNA molecule are said to be
   antiparallel. What does antiparallel mean?gt
4. During cell division, DNA molecules are
   replicated in a semiconservative manner. What
   happens to the original DNA molecule during
   semiconservative replication?

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4.2 Sources of DNA
In nature, DNA is made in cells.
Mammalian Cell Culture

• Growing mammalian cells in culture is more
  challenging than growing bacterial cells
• Mammalian cells are grown in a broth culture

Viral DNA

• Viruses are classified according to the type of
  cell they attack
• Bacterial (bacteriophages)
• Plant
• Animal

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Prokaryotic DNA
Bacterial Operon. An operon contains the
controlling elements that turn genetic expression
ON and OFF.
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Bacterial Cell Culture
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Eukaryotic DNA
Eukaryotic Gene. Eukaryotic genes have a
promoter to which RNA polymerase binds, but they
do not have an operator region.
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Vocabulary

• Medium a suspension or gel that provides the
  nutrients (salts, sugars, growth factors, etc.)
  and the environment needed for cells to survive
  plural is media
• Lysis the breakdown or rupture of cells
• R plasmid a type of plasmid that contains a
  gene for antibiotic resistance
• Transformed refers to those cells that have
  taken foreign DNA and started expressing the
  genes on the newly acquired DNA
• Vector a piece of DNA that carries one or more
  genes into a cell, usually circular as in plasmid
  vectors
• Operon a section of prokaryotic DNA consisting
  of one or more genes and their controlling
  elements
• RNA polymerase an enzyme that catalyzes the
  synthesis of complementary RNA strands from a
  given DNA strand
• Promoter the region at the beginning of a gene
  where RNA polymerase binds the promoter
  promotes the recruitment of RNA polymerase and
  other factors required for transcription
• Operator a region on an operon that can either
  turn on or off expression of a set of genes
  depending on the binding of a regulatory molecule
• Beta-galactosidase an enzyme that catalyzes the
  conversion of lactose into monosaccharides

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Vocabulary

• Agar solid media used for growing bacteria,
  fungi, plant, or other cells
• Media preparation the process of combining and
  sterilizing ingredients (salts, sugars, growth
  factors, pH indicators, etc.) of a particular
  medium
• Autoclave an instrument that creates high
  temperature and high pressure to sterilize
  equipment and media
• Enhancer a section of DNA that increases the
  expression of a gene
• Intron the region on a gene that is transcribed
  into an mRNA molecule but not expressed in a
  protein
• Exon the region of a gene that directly codes
  for a protein it is the region of the gene that
  is expressed
• Transcription factors molecules that work to
  either turn on or off the transcription
  eukaryotic genes
• Histones nuclear proteins that bind to
  chromosomal DNA and condense it into highly
  packed coils
• Nonpathogenic not known to cause disease
• Bacteriophages viruses that infect bacteria
• Gene therapy the process of treating a disease
  or disorder by replacing a dysfunctional gene
  with a functional one

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4.2 Review Questions

1. Plasmids are very important pieces of DNA. How do
   they differ from chromosomal DNA molecules?
2. Bacteria cell DNA is divided into operons.
   Describe an operon using the terms promoter,
   operator, and structural gene.
3. Describe the human genome by discussing the
   number and types of chromosomes, genes, and
   nucleotides.
4. What is gene therapy? Cite an examples of how it
   can be used.

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4.3 Isolating and Manipulating DNA

1. Identification of the molecule(s)
2. Isolation of the instructions (DNA
   sequence/genes) for the production of the
   molecule(s)
3. Manipulation of the DNA instructions
4. Harvesting of the molecule or product, testing
   it, and marketing it

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Recombinant DNA Technology
Methods to create new DNA molecules
Site-Specific Mutagenesis
Process of including changes (mutagenesis) in
certain sections (site-specific) on a particular
DNA code
Gene Therapy
Process of correcting faulty DNA codes that cause
genetic diseases and disorders
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Vocabulary

• Bioremediation the use of bacteria or other
  organisms to restore environmental conditions
• Site-specific mutagenesis a technique that
  involves changing the genetic code of an organism
  (mutagenesis) in certain sections (site-specific)

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4.3 Review Questions

• Genetic engineering by any method requires
  certain steps. Put the following steps in the
  correct order
• isolation of the instructions (DNA
  sequence/genes)
• harvest of the molecule or product then
  marketing
• manipulation of the DNA instructions
• identification of the molecule to be produced
• What naming designation is used with
  recombinant products made through genetic
  engineering?
• What is the smallest change in a DNA molecule
  that can occur after site-specific mutagenesis?
  What effect can this change have?
• What gene has been the target of CF gene therapy?
  What does this gene normally do?

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4.4 Using Gel Electrophoresis to Study Gene
Molecules
Components of Gel Electrophoresis

• Powdered agarose
• Boiling buffer solution

Agarose Gel Concentrates
Most commonly used when separating pieces of DNA
no smaller than 50 bp and no larger than 25,000 bp
Gel Stains
The gel is run until molecules of different
sizes are thought to have completely separated.
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Agarose Gel Tray. Gel trays differ depending on
the manufacturer. Each has some method of sealing
the ends so that liquid agarose can mold into a
gel. Some gel trays, such as those made by Owl
Separation Systems, make a seal with the box, so
casting a gel is simple. Other trays require
masking tape on the ends to make a mold. Still
others, like the one shown here, have gates that
screw into position up for pouring the gel and
down for running the gel.
Molecules in a Gel Box. If negatively charged
molecules are loaded into the wells and run on
the gel, the smaller ones run faster and farther
than the larger ones toward the positive
electrode. This is because smaller molecules pass
more easily through the tiny spaces of the gel
network.
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Vocabulary

• Gel electrophoresis a process that uses
  electricity to separate charged molecules, such
  as DNA fragments, RNA, and proteins, on a gel
  slab
• Agarose a carbohydrate from seaweed that is
  widely used as a medium for horizontal gel
  electrophoresis
• Polyacrylamide a polymer used as a gel material
  in vertical electrophoresis used to separate
  smaller molecules, like proteins and very small
  pieces of DNA and RNA
• Ethidium bromide a DNA stain (indicator) glows
  orange when it is mixed with DNA and exposed to
  UV light abbreviated EtBr
• Methylene blue a staining dye/indicator that
  interacts with nucleic acid molecules and
  proteins, turning them to a very dark blue color
• High through-put screening the process of
  examining hundreds or thousands of samples for a
  particular activity

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4.4 Review Questions

1. Agarose gels can be used to study what size of
   DNA fragments?
2. If agarose gel material is labeled 1, what does
   the 1 refer to?
3. What causes molecules to be separated on an
   agarose gel?
4. Name two common DNA stains that are used to
   visualize DNA on agarose gels.

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Questions and Comments?