Unit 3: Introduction to Studying DNA


Dec 14, 2012 (4 years and 4 months ago)


Describe the structure and function of DNA and explain how
proteins are made

Differentiate between eukaryotic and prokaryotic cells and
chromosome 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 type using sterile

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 discuss how the
characteristics of molecules affect their migration through a gel

The manipulation of genetic
information, specifically the
deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) codes, is the
center of most biotech research and

The genetic information within cells is
stored in

Before we get into DNA lets review

A Review of Cell Structure

Online activity on cells:

There are
two major types of cells

Prokaryotic Cells

Eukaryotic Cells

Key Terms to Remember!

Plasma Membrane

layer structure
of lipids and proteins that surrounds the
outer surface of cells


inner contents of a cell between
the nucleus and plasma membrane


structures in the cell that
perform specific functions

Prokaryotic Cells
(include bacteria)

No nucleus and no organelles

Have a nucleus and many organelles




Endoplasmic reticulum

Golgi apparatus

Vero cells

are a cell line which is widely used to make
vaccines. The cell line was derived from kidney epithelial cells of
the African Green Monkey. The cell line was established in 1962 by
Japanese scientists. Vero cells are susceptible to a broad range of
viruses so they are used to develop vaccines diseases associated
with those viruses. One of the vaccines for which Vero cells have
been used in recent years is the vaccine against influenza ("the

HeLa Cells

cells are the first immortal cell line, or a cell line that
continues to reproduce and "live" outside the human body. These
cells have been used in cell research in projects that have benefited
mankind around the world. The original cells were taken from
cancerous cervical tumor from a poor African
American woman
named Henrietta Lacks who died from cervical cancer in 1951.

Escherichia coli

(commonly abbreviated
E. coli)

is a gram
negative, rod shaped bacteria that is commonly found in the lower
intestine of warm blooded organisms.

Most E. coli strains are harmless, but some can cause serious food
poisoning in humans, and are occasionally responsible for product
recalls. The harmless strains are part of the normal flora of the gut.

E. coli are not always confined to the intestine, and their ability to
survive for brief periods outside the body makes them an ideal
indicator organism to test environmental samples for fecal

The bacteria can also be grown easily and its genetics are
comparatively simple and easily manipulated or duplicated
through a process of metagenetics, making it one of the best
studied prokaryotic model organisms, and an important species in
biotechnology and microbiology.

Chinese hamster ovary cells or CHO

are cells that have been derived from the ovary of
the Chinese hamster. In year 1957, scientist T.
Puck at Dr. George Yerganian's laboratory at the
Boston Cancer Research Foundation used a female
Chinese hamster to extract this cell line. Today, it
is a widely used mammalian cell line in biological
research since its introduction in the year 1960.

One of the characteristics of CHO cells is that it
requires the amino acid “Proline” for its growth
and it’s an adherent monolayer cell line.

A rapid growing cell line with excellent ability to
express recombinant protein makes it the cell line
of choice in experiments relating to gene
expression, genetics, toxicity screening and
nutritional studies.


are particles of
nucleic acid, protein, and in
some cases, lipids.

Viruses can reproduce
by infecting living cells

Viruses differ widely in
terms of size and structure.

As different as they are, all
viruses have one thing in
common: They enter living
cells and, once inside, use
the machinery of the infected
cell to produce more viruses.

virus's protein coat

called its

The capsid includes
proteins that enable a virus
to enter a host cell.

The capsid proteins of a
typical virus bind to
receptors on the surface of
a cell and “trick” the cell
into allowing it inside.

Once inside, the viral genes
are expressed.

The cell transcribes and
translates the viral genetic
information into viral
capsid proteins.

Sometimes that genetic
program causes the host
cell to make copies of the
virus, and in the process
the host cell is destroyed

Because viruses must bind precisely to
proteins on the cell surface and then use a
host's genetic system, most

Viruses are highly specific to the cells they infect.

Plant viruses infect plant cells

Most animal viruses infect only certain related
species of animals

Bacterial viruses infect only certain types of
Viruses that infect Bacteria are called

To View how a virus attacks watch this video!

Learn the Basics Website

Building block of DNA is the

Each nucleotide is composed of

Pentose (5

called deoxyribose



nitrogenous base

The nitrogenous bases are the interchangeable
component of a nucleotide

Each nucleotide contains one base

Adenine (A), thymine (T), guanine (G) or
cytosine (C)

Nucleotides are joined together to
form long strands of DNA and each
DNA molecule consists of two strands
that join together and wrap around
each other to form a
double helix

in a strand

are held
together by
phosphodiester bonds

Each strand has a

a 5’ end
and a 3’ end

The two strands of a DNA molecule are held together by
hydrogen bonds

Formed between complementary base pairs

Adenine (A) pairs with thymine (T)

Guanine (G) pairs with cytosine (C)

The two strands are

because their polarity is
reversed relative to each other

Chromosome Structure


highly coiled and tightly
condensed package of DNA and proteins

Occurs only during DNA replication


strings of DNA and DNA
proteins called histones

State of DNA inside the nucleus when the cell is
NOT dividing

Most human cells have two sets (pairs) of
23 chromosomes, or 46 chromosomes total

homologous pairs


chromosomes 1

Sex chromosomes

chromosome pair # 23

X and Y chromosomes


(sex cells)

contain a single set of
23 chromosomes (haploid number, n)

DNA Replication

Cells divide by a process called

Sex cells divide by a slightly different process called


One cell divides to form two daughter cells, each with
an identical copy of the parent cell DNA

In order to accomplish this, the DNA of the parent cell
must be copied prior to mitosis

DNA undergoes
Semiconservative Replication

Replication occurs in such a manner that, after
replication, each helix contains one original
(parental) DNA strand and one newly synthesized
DNA strand

Unwinding the DNA


enzyme breaks the hydrogen bonds
holding the two DNA strands together;
“unzips” DNA

DNA binding proteins hold the strands apart

Separation of strands occurs in regions called
origins of replication

Adding short segments of RNA


enzyme adds RNA primers

RNA primers start the replication process

3. Copying the DNA

DNA polymerase enzyme binds to the

RNA primers

Uses nucleotides to synthesize
complementary strands of DNA

Always works in one direction

5’ to 3’

Occurs only in

RNA polymerase

unwinds DNA helix and
copies one strand of DNA into RNA

Binds to a


Copies DNA in a 5’ to 3’ direction into RNA

Uses nucleotides

, guanine, and

At end of gene, RNA polymerase
encounters the
termination sequence

RNA polymerase and newly formed
strand of RNA are released from DNA

RNA strand is called a
messenger RNA

Multiple copies of mRNA are transcribed
from each gene during transcription

The mRNA then gets processed

Initial mRNA produced is the primary

Immature and not fully functional

A series of modifications before
primary transcripts are ready for
protein synthesis

RNA splicing


Addition of a 5’ cap

mRNA is read during a process called

Translation takes place in the cytoplasm

Works in three nucleotide units of mRNA

Each codon codes for a single amino acid

One amino acid may be coded for by more
than one codon

Start codon (AUG)

Stop codons

There are three different types of RNA
involved in the translation process


exact copy of the gene; carries
the genetic code from nucleus to the


component of ribosomes, the
organelles responsible for protein


transports amino acids to



small ribosome subunit binds to 5’
end of mRNA

Moves along the mRNA until the start codon is
found (AUG)



tRNAs, carrying the correct
amino acid, enter the ribosome, one at a time,
as the mRNA code is read



ribosome encounters the stop

Newly formed protein is released

Many Biotech efforts modify DNA
molecules with the goal of affecting
protein production

In humans, about 40,000 genes are needed
for an organism to function

A typical cell synthesizes more than 2000
different kinds of proteins and hundreds
or thousands of copies of these proteins are
usually needed at any given time

If you multiply these numbers by the
hundreds of types of cells we have in our
body, the numbers reach into the millions

The entire sum of DNA in a cell is variable
from organism to organism, but every cell
within an organism, except sex cells, has
the same genome.

Even though there are different
quantities of DNA in cells from different
organisms, the DNA itself is virtually


Haemophilus influenza: childhood ear infections

Mycoplasma genitalium: Free living bacterium

Caenorhabditis elegans: Free
living roundworm

Homo sapiens: humas

#of Genes





Size (bp)





All DNA molecules are made of A,T,C,G

Virtually all DNA molecules form a double

The nucleotides connect to one another via
phosphodiester bonds between the sugar and

Hydrogen bonds hold each base to its
complementary base creating the two sides of the
DNA molecule

The amount of A is always equal to the
amount of T

The amount of G is always equal to the
amount of C

In prokaryotic cells, the DNA is floating in
the cytoplasm.

It typically contains only one, long, circular
DNA molecule (chromosome)

It is usually folded in on itself and only contains
several thousand genes (compared to a human
who has around 40,000 genes)

Some bacteria
contain small rings of
DNA that are outside
of the “chromosome”
floating in the
cytoplasm called

Plasmids only
contain a few genes
(5 to 10) that
usually code for
proteins that offer
some additional
characteristics that
may be needed only
under extreme

The most common type of plasmids are
R plasmids

R plasmids contain antibiotic resistance genes

These genes allow the bacteria to survive exposure to
antibiotics that would normally kill them

Bacteria can transfer genetic information between
themselves by a process called

Transferring plasmids give bacteria a way of
“evolving” by gaining new and better characteristics

Because plasmids are pieces of DNA that
can accept, carry, and replicate (clone)
other pieces of DNA, they are often used as

A Vector is a vehicle used to transfer the genetic
material such as DNA sequences from the
donor organism to the target cell of the
recipient organism.


Just like other enzymes, restriction enzymes show specificity
for certain substrates (DNA)

They work by cutting the phosphodiester bond (in the sugar
phosphate backbone) that joins adjacent nucleotides in a
DNA strand.

The cut DNA within a specific sequence of bases called a
recognition sequence

restriction site

Modifications of DNA can be as simple as
changing a single base

Changing DNA sequences may alter the
production of some proteins in a cell or

New proteins may also be created

Genetic Engineering:
the production of
rDNA molecules and their insertion into

rDNA: (recombinant DNA) :
pieces of
DNA that have been cut and then pasted
back together

These technologies are the methods
to create new DNA molecules by piecing
together different DNA molecules

When cells accept the
and start
expressing the new genes (by making the
new proteins) they are considered
genetically engineered.

The names of proteins produced in this
way are written with an “r” in front of

Example : rInsulin

specific mutagenesis
refers to the process
of inducing changes (mutagenesis) in certain
sections (site
specific) of a particular DNA

The changes in the DNA code are usually
accomplished through the use of chemicals,
radiation, or viruses.

Sometimes site
specific mutagenesis is “directed”
meaning a scientist is trying to make certain changes
in a protein’s structures that will translate into an
improved function.

Example: Subtilisin is an enzyme that is added to laundry
detergent to remove proteinacous stains (such as blood or
gravy) This protein was made from altering the protein
subilisin found in fungi. The alteration made the protein
able to function in an alkaline soln. such as laundry det.

Gene Therapy
is the process of correcting
faulty DNA codes that cause genetic
diseases and disorders.

The most common way to conduct gene
therapy is to use a virus to carry a normal gene
into cells containing defective genes (basically
gene replacement)

This therapy is currently being used for Cystic
Fibrosis, Parkinson's, diabetes, and some cancers

Gel Electrophoresis uses electricity to
molecules using a gel slab.

By using electrophoresis, researchers can easily
separate and visualize charged molecules, such as
DNA fragments, RNA and proteins

The two most common types of gels are

(isolated from sea weed) and

are used to separate smaller molecules such as
proteins and very small pieces of DNA or RNA)

Agarose gels are commonly made with concentrations
ranging from 0.6% to 3% agarose in buffer (the more
concentrated the more “straining effect” the gel will

Most common gel for DNA fragment separation is .8%



The two types of stain that can be used to
visualize fragments on a gel are

Ethidium Bromide (EtBr): this is the most
common DNA gel stain; it glows orange when
mixed with DNA and exposed to UV light.

EtBr is a mutagen!!! (This means it causes cancer!)

Methylene blue: will bond with the nucleic acid
molecules, turing them a dark blue color

Methylene blue is not as sensitive as EtBr so the
banding pattern will be harder to see!

The body possesses three lines of
defense to prevent and fight off
intrusions by pathogens

The first two lines are non

The third layer is the body’s specific
immune system.

Specific immune responses are
tailored to the type of invading

Specific immune responses are triggered by


Antigen (Antibody Generator): proteins or other
molecules produced by pathogens

The key players in the specific immune
response are the
dendritic cells, macrophages,
and small white blood cells called B
lymphocytes (B cells) and T lymphocytes (T

Phagocytic macrophages and dendritic cells break
down pathogens and display antigenic fragments
from the pathogens on the surface of their cell

The B cells and T cells circulate through the body in
the blood and lymph looking for these displayed
antigenic fragments.

When the T cells see the displayed
antigenic fragment, they stimulate specific
B cells to reproduce and generate

designed against the specific
structure of the antigen that was

Antibodies (immunoglobulins):
a group of
serum proteins that are found in the
bloodstream that have a y
shaped structure but
have different antigen binding sites at their

Antigen binding sites are designed to fit the
shape of specific antigens

Antibodies bind to antigens like a lock
and key forming an antigen

When the Ab/Ag complex forms it marks
the invading organism/antigen for
destruction by phagocytic cells

It also stimulates additional immune

ELISA: Enzyme Linked Immunosorbant Assay

This assay is based on the principle that antibodies
are produced in the response to pathogens

The antibodies attach to their antigen targets with
great specificity to form Ag/Ab complexes.

There are two types of ELISA tests

Indirect ELISA:

Direct ELISA: