Biotechnology Chapter Four Lecture- DNA

Introduction to
Studying DNA
Chapter 4

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

Learning Outcomes






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

The manipulation of
genetic information,
DNA and RNA
codes, is at the
center of most
biotechnology
research and
development.

Section 4.1- DNA
Structure and Function

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.

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.

Phosphodiester
bonds
connect sugar
molecules.
The phosphate
groups have a
negative charge.

Nitrogenous bases are purines (two rings)
or pyrimidines (single ring)

Nitrogenous bases from
each strand bond to
each other in the center
through H-bonds.
A with T (2 H-bonds)
G with C (3 H-bonds)
Always purine with
pyramidine giving the
double strand
consistent width

The H-bonds are rather
weak; therefore, the
two strands of DNA
separate easily in high
temperatures.
G/C rich DNA will
separate at slightly
higher temps due to
having more H-bonds

Similarities in DNA Molecules
Among Organisms
All DNA molecules are composed of four nucleic monomers
Adenosine deoxynucleotide (A), Cytosine deoxynucleotide (C),
Guanosine deoxynucleotide (G), and 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

due to base pairing

Among Organisms

DNA is antiparallel-

✦nucleotides in each strand are oriented
in opposite direction of the other strand

✦one strand is 3’ to 5’ the other is 5’ to 3’
4.

Among Organisms

10 base pairs / turn-The double helix has
a regular shape which is recognized by
other molecules.
4.

DNA undergoes
semiconservative replication

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.

Variations in DNA Molecules

•

The number of DNA strands in the
cells of an organism

•

The length of the DNA strands

•

The number and type of genes and
noncoding regions

•

The shape of the DNA strands

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 or two
nitrogen-containing rings; forms the critical hydrogen
bonds between opposing strands of a double helix
• Base pair – the 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

Vocabulary
• 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
involves 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

Vocabulary
• 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

4.1 Review Questions

2.

1. Describe the relationship between genes, mRNA,
and proteins.

3.

2. Name the four nitrogen-containing bases found in
DNA molecules and identify how they create a base
pair.

4.

3. The strands on a DNA molecule are said to be
“antiparallel.” What does antiparallel mean?

5.

4. During cell division, DNA molecules are replicated
in a semiconservative manner. What happens to the
original DNA molecule during semiconservative
replication?

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

4.2 Sources of DNA


Bacterial Cell Culture

Some bacteria prefer a liquid medium (nutrient broth),
and some prefer a solid medium (nutrient agar).

4.2 Sources of DNA



Agar plates contain nutrient broth gelled
with agar protein.

4.2 Sources of DNA



All media must be prepared under sterile
conditions.

4.2 Sources of DNA



Introduction of cells must be done under
sterile conditions to ensure purity.

4.2 Sources of DNA



An autoclave
sterilizes equipment
and solutions with
high pressure and
temp.

4.2 Sources of DNA



Sterile technique is
an important lab skill
for a microbiologist.

4.2 Sources of DNA



Sterile technique is
doing something
without
contamination by
unwanted organisms
or their spores.

Bacterial genes
In prokaryotes the DNA is ﬂoating in the cytoplasm.

Bacterial genes
There is one large, twisted, circular chromosome
that contains essential genes.

Bacterial genes
There may be plasmids- small circular DNA
molecules with non-essential but often
advantageous genes

Bacterial genes
Plasmids are transferred between bacteria in
nature and in the lab allowing easy transfer of
genes between prokaryotes.

Bacterial genes
Plasmids are good vectors for gene recombination.
A vector is a vehicle used to transfer genetic material.

Prokaryotic DNA regulation is simple

Bacterial Operon
An operon contains the controlling elements that
turn genetic expression ON and OFF.


RNA polymerase attaches to the promoter.


Attachment of an inhibitor to the operator
region will block production turning the
gene off.

-the lac operon in E.coli

Genes must be transferred
along with appropriate
regulatory sequences.

Eukaryotic DNA

Eukaryotes have multiple chromosomes

Eukaryotic DNA

Each chromosome contains a single linear
(very long) DNA molecule wrapped around
histone proteins.

Eukaryotic DNA

Tightly coiled chromosomes hide genes.
Some unwinding from histones must occur
for gene expression.

Eukaryotic DNA

There is much spacer DNA that does not
code for anything.

Eukaryotic DNA

Eukaryotic Gene. 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.

Eukaryotic DNA

RNA polymerase moves down the DNA molecule
until it reaches a structural gene which is
transcribed until a termination sequence is
reached.

Eukaryotic DNA

The transcribed RNA molecule has introns
(noncoding) removed and exons spliced together
which leave the nucleus as mRNA.

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 – the cells that have taken foreign DNA
and started expressing the genes on the newly
acquired DNA
•

Vocabulary
• 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
•

Vocabulary
• 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

Vocabulary
• Agar – a 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
• Silencer – a section of DNA that decreases the expression
of a gene

Vocabulary
• Transcription factors – molecules that work to
either turn on or off the transcription eukaryotic
genes
• 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
• Histones – the nuclear proteins that bind to
chromosomal DNA and condense it into highly
packed coils

Vocabulary

• Nonpathogenic – not known to cause
disease
• Bacteriophages – the viruses that infect
bacteria
• Gene therapy – the process of treating a
disease or disorder by replacing a
dysfunctional gene with a functional one

.

.

.

.

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.

4.3 Isolating and Manipulating DNA
Basic steps of genetic engineering

1. Identiﬁcation of the molecule(s)What do you want to produce?
2. Isolation of the instructions (DNA
sequence/genes) for the production
of the molecule(s)

4.3 Isolating and Manipulating DNA
Basic steps of genetic engineering

3. Manipulation of the DNA instructions
-change the DNA
or
-move it to a new organism
4. Harvest the molecule or product and
test it
To make a proﬁt, the product must also be marketed.

Recombinant DNA Technology

Recombinant DNA is made by combining
different DNA molecules.
Recombinant DNA and the
proteins produced from it have an
“r” before their name.
rhInsulin =
recombinant human insulin

Recombinant DNA Technology
http://www.dnalc.org/view/15255-Producing-humaninsulin-using-recombinant-DNA-Walter-Gilbert.html

http://www.youtube.com/watch?
v=mhvARWPS1zM

Site-Speciﬁc Mutagenesis

Process of inducing changes
(mutagenesis) in certain sections (sitespeciﬁc) on a particular DNA code

Random!

Expose cells to chemicals, viruses,
or radiation causing random
mutations.
Screen mutants for desirable traits.

v=DjGLUnJSRYs

directed!!

http://video.google.com/videoplay?
docid=5044846172948251835

Gene Therapy

Process of correcting faulty DNA
codes that cause genetic diseases
and disorders

Gene Therapy

and disorders
Manipulation must occur in mature
multicellular organism

Gene Therapy

and disorders
Manipulation must occur in mature
multicellular organism
Viruses are used to insert functional
genes.

v=gl2miunHTRI&feature=related

Vocabulary

• Site-specific mutagenesis – a
technique that involves changing
the genetic code of an organism
(mutagenesis) in certain sections
(site-speciﬁc)


1. Genetic engineering by any method requires certain
steps. Put the following steps in the correct order:


isolation of the DNA sequence



harvest of the molecule or product




manipulation of the DNA instructions





identiﬁcation of the molecule to be produced





identiﬁcation of the molecule to be produced
2. What “naming” designation is used with
recombinant products made through genetic
engineering?


3. What is the smallest change in a DNA
molecule that can occur after site-speciﬁc
mutagenesis? What effect can this
change have?
4. What gene has been the target of CF
gene therapy? What does this gene
normally do?
CFTR gene = cystic ﬁbrosis transmembrane conductance regulator

4.4 Using Gel Electrophoresis to Study
Gene Molecules

Gene Molecules

DNA is negatively charged and moves
towards the + end of an electric ﬁeld.

Gene Molecules

Moving molecules are separated by size
in a “gel”. Smaller pieces travel farther.

Gene Molecules

Moving molecules are separated by size
in a “gel”. Smaller pieces travel farther.
DNA is chopped up by restriction
enzymes before loading in gel.

Gene Molecules

agarose gel- Medium to large DNA
pieces (500bp - 25,000bp)
Poly-acrylamide (aka PAGE)- smaller
stuff like protein, RNA, or small DNA

Components of Agarose Gel
Electrophoresis

Powdered agarose + boiling TRIS buffer
solution is made to speciﬁed concentration

Electrophoresis

0.8% is appropriate for most restriction
enzyme digested DNA

Electrophoresis

0.8% is appropriate for most restriction
enzyme digested DNA
3% agarose gels would be much slower
running

Electrophoresis

A loading dye is used to visualize the
movement of molecules.

Electrophoresis

A set of DNA fragments of known size
are also run for comparison.

Gel Stains

Methylene blue makes the DNA
visible.

Gel Stains

Ethidium
bromide (EtBr)
makes DNA glow
orange in UV
light.
More sensitive
More
hazardous

Gel trays differ
depending on the manufacturer.
Each has some method of sealing
the ends so that liquid agarose
can mold into a gel.
Agarose Gel Tray.

For the gel box to
conduct electricity, the solution in the gel box
must contain ions.
Sodium chloride (NaCl) solution can be used,
but other salts, such as TRIS or lithium,
dissolved in water (called a “running buffer”),
are better for conducting electricity.
Molecules in a Gel Box.

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

Vocabulary

• 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


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?


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.

Biotechnology Chapter Four Lecture- DNA

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