CHAPTER 4:
INTRODUCTION TO STUDYING DNA
Introduction to Biotechnology, BIOL1414
Austin Community College, Biotechnology Dept
LEARNING OUTCOMES
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Describe the structure and function of DNA and explain
the process by which it encodes for proteins
Describe how DNA is replicated in the cell
Differentiate between eukaryotic and prokaryotic
chromosomal structure and explain how this difference
impacts gene regulation in the two cell types
Describe the process of gel electrophoresis and explain
how the characteristics of molecules affect their
migration through a gel
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DNA STRUCTURE & FUNCTION

The manipulation of genetic information, DNA
and RNA codes, is at the center of most
biotechnology research and development.
http://www.historyforkids.org/scienceforkids/biology/cells/dna.htm
DNA DISCOVERY (VISIT DNAI.ORG)
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Miescher – identified a nuclear substance he called nuclein
Griffith – performed the first transformation
Avery, McCarty, and Macleod – identified Griffith’s transforming
factor as DNA
Chargaff – proved that the percentage of the DNA bases adenine
always equaled thymine and guanine always equaled cytosine
Wilkins, Franklin, Watson & Crick – demonstrated the structure
of DNA
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.
SIMILARITIES IN DNA
MOLECULES AMONG ORGANISMS
1.
2.
3.
4.
5.
6.
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 semi-conservative
replication
VARIATIONS IN DNA MOLECULES
 DNA
from organism to organism varies in:

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 (circular
vs linear)
DNA STRUCTURE
 The
nucleotides in one
chain of the helix face one
direction, while those in the
other strand face the other
direction : ANTIPARALLEL
 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
Hydrogen-bonds.
STRUCTURE OF DNA - NUCLEOTIDES
•
•
•
Deoxyribose
Sugar
Phosphate
Nitrogen
Base
STRUCTURE OF DNA - NUCLEOTIDES
 Purines
– double ring
 Pyrimidines
– single ring
STRUCTURE OF DNA
Nucleic Acid Overview
WHAT IS A GENE?
A
gene is a sequence of nucleotides that
provides cells with the instructions to
synthesize a specific protein.
 Note, that not all genes produce protein!
 Most
genes are 1000-4,000 nt long and
encode for a particular trait
 Note, some traits are encoded by one
gene, but most are determined by
multiple genes!
WHAT IS A GENOME?
 DNA
contains the instructions for life – genes
 All the DNA in an organism’s cells is called a
genome
 The human genome contains over 3 billion bases
and 23,000 genes.
 The study of genomes is called genomics
 Genomics
is an exciting career area!
STRUCTURE OF DNA – KARYOTYPE ANALYSIS
DNA REPLICATION
 When
DNA makes an exact copy of itself
 Growth & Development
 Replace aging/damaged cells
DNA Replication Animation
DNA REPLICATION
 DNA
replicates in a
semi-conservative
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.
DNA Replication
DNA REPLICATION
 The
first step in
DNA replication is
for the enzyme,
helicase, to unzip
the double
stranded DNA
molecule.
DNA REPLICATION
 Proteins
hold the two strands apart.
 An RNA primer lays down on each strand of
DNA.
DNA REPLICATION
 DNA
polymerase extends the primer by adding
complementary nucleotides.
 DNA polymerase can only extend in the 5’ → 3’
direction
DNA REPLICATION
 Leading
strand follows helicase.
 Lagging strand must wait for replication fork to
open and therefore forms discontinuous Okazaki
fragments.
 Ligase seals the nicks in the DNA backbone
between the Okazaki fragments.
helicase
LET’S PUT IT ALL TOGETHER
Click on the animation below.
 Select the button for the “whole picture”.

DNA Replication Animation
SOURCES OF DNA

In nature, DNA is made in cells.
Mammalian Cell Culture
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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:
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Bacterial (bacteriophages)
Plant
Animal
PROKARYOTIC
DNA
Gene Expression in prokaryotes is much more simple
than eukaryotes
 An operon contains the controlling elements that turn
genetic expression ON and OFF.
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THE LAC OPERON
LET’S PUT IT ALL TOGETHER
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Click on the animation below.
Animation of lac operon
Video of lac operon
BACTERIAL CELL CULTURE
 Some
bacteria grow well in liquid medium –
broth
 Some bacteria prefer solid medium – agar
 Some grow on both for different purposes
http://cals.arizona.edu/main/spotlight/how-microbes-take-out-trash
http://www.sciencelearn.org.nz/Contexts/Enviro-imprints/Sci-Media/Images/E.coli
EUKARYOTIC DNA
Eukaryotic genes have a promoter to which RNA
polymerase binds, but they do not have an operator
region.
 Transcription factors may bind at enhancer
regions and increase gene expression.

MAMMALIAN CELL CULTURE
Growing mammalian cell culture is far more
challenging and expensive than bacterial cell culture
 Typically grown in broth culture in special flasks
 Specific media designed to have all special nutrients of
that cell type
 Special indicators can be added to monitor grown (such
as phenol red)
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ISOLATING AND
MANIPULATING DNA
1.
2.
3.
4.
Identification of molecules for our benefit
– Insulin for example
Isolation of DNA (gene)
Manipulation of DNA – insert into a
different organism to produce the gene
product
Harvest to the molecule of interest from
the host organism
USING GEL ELECTROPHORESIS TO
STUDY BIOLOGICAL MOLECULES
Gel Electrophoresis:
 Most commonly used when separating pieces of
DNA no smaller than 50 bp and no larger than
25,000 bp
 The gel is “run” until molecules of different sizes
are thought to have completely separated.
Components of Gel Electrophoresis
 Powdered agarose
 Boiling buffer solution
 Running buffer
 DNA stain
 Sample load buffer
GEL ELECTROPHORESIS

Electrophoresis is a molecular technique that can
(depending on application) separate nucleic acids
and proteins based on:
Size
and/or
+-+Charge+-+
DNA AGAROSE GEL ELECTROPHORESIS
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Click here for animation on Agarose gel
electrophoresis!
Click here to watch a video on Agarose gel
electrophoresis!
AGAROSE GEL ELECTROPHORESIS
 DNA
is analyzed by size alone on agarose
gel electrophoresis.
 DNA is a negatively charged molecule and
therefore is attracted to positive charges.
AGAROSE GEL ELECTROPHORESIS
 Agarose
provides a matrix through which
DNA molecules migrate.
Larger molecules move through the matrix slower
than small molecules
 The higher the concentration of agarose, the better
the separation of smaller molecules

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.
DNA AGAROSE GEL
ELECTROPHORESIS
DNA AGAROSE GEL
ELECTROPHORESIS
DNA AGAROSE GEL
ELECTROPHORESIS
For the gel box to conduct electricity and establish
an electric field with a positive end (red wire) and a
negative end (black wire), the solution in the gel box
must contain ions.
 The smallest molecules run fastest thru the gel

DNA AGAROSE GEL ELECTROPHORESIS
 DNA
fragments
separate according
to size.
 Smaller
fragments run
faster through
the agarose mesh
AGAROSE GEL
ELECTROPHORESIS
 How
1.
2.
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4.
5.
to make an agarose gel:
Weigh out a specified amount of
agarose powder.
Add the correct amount of buffer.
Dissolve the agarose by boiling the
solution.
Pour the gel in a casting tray.
Wait for the gel to cool and solidify
AGAROSE GEL ELECTROPHORESIS
 How
to make an agarose
gel:
6. Place gel in chamber and
cover with buffer
7. Add loading dye to the
sample
8. Load sample on to the
gel.
9. Run at constant voltage
AGAROSE GEL ELECTROPHORESIS
 How
to make an agarose
gel:
10. Stain the gel –
(Ethidium Bromide,
SYBR green, methylene
blue…)
11. Capture an image of the
gel
12. Analyze results
GENOMIC DNA ANALYSIS ON AGAROSE GEL
Genomic DNA isolated from Iris Plants .
 Not RNase treated.
 1% agarose gel, 100V, 60min
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M
10,000 bp
1
2
3
genomic DNA
3,000 bp
1,000 bp
rRNA
ANALYZING RNA

RNA is another very important nucleic acid
commonly isolated and analyzed in a biotechnology
lab
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RNA provides the link between the genetic information
stored in DNA and the expression of that information
through protein synthesis.
Differences in RNA and DNA structure:
RNA has deoxyribose
 RNA has many different 3-D structures
 RNA has A, U, G, C nucleotides
 RNA is involved in many different functions including
transcription, translation and gene regulation to name
a few!
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Learn More here
DENATURING AGAROSE GEL - RNA
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Unlike DNA, RNA comes in many different 3-D
shapes that affect it’s migration through an agarose
gel.
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If the particle is small and tightly packed it migrates
further/faster than a long cylinder shape particle of the
same size.
In order to accurately determine and compare sizes
of RNA molecules you must first denature the RNA
into a linear form.
The equipment is the same as with DNA analysis,
but the buffer used is denaturing – either
formaldehyde, or glyoxal buffers work well
HOW TO PREPARE AN AGAROSE GEL
How to set up DNA Agarose
Gel Electrophoresis
Click here!
How to set up RNA Agarose
Gel Electrophoresis
Click here!
QUESTIONS AND COMMENTS?
REVIEW QUESTIONS
Your Turn!
Put your name at the top of a sheet of paper, answer
these questions and hand in:
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 “anti-parallel.”
What does anti-parallel mean?
4.
During cell division, DNA molecules are replicated in a semiconservative manner. What happens to the original DNA
molecule during semi-conservative replication?
5.
How are small strands of DNA separated in a typical
biotechnology lab? What equipment is needed?
REFERENCES
1.
Biotechnology: Science for the New Millennium.
2012. Ellyn Daugherty.